Compositions including triciribine and trastuzumab and methods of use thereof

ABSTRACT

This application relates to combination therapies including triciribine and related compounds and trastuzumab or a salt thereof and compositions with reduced toxicity for the treatment and prevention of tumors, cancer, and other disorders associated with abnormal cell proliferation.

The present application is a continuation of U.S. patent applicationSer. No. 13/453,778, filed Apr. 23, 2012, which issued as U.S. Pat. No.8,623,834, which is a continuation of U.S. patent application Ser. No.12/118,848, filed May 12, 2008, which is abandoned, and is acontinuation-in-part of U.S. patent application Ser. No. 11/096,082,filed Mar. 29, 2005, which issued as U.S. Pat. No. 8,435,959, whichclaims the benefit of U.S. provisional patent application No.60/557,599, filed Mar. 29, 2004, each of which is incorporated herein byreference.

ACKNOWLEDGEMENT OF GOVERNMENT SUPPORT

This invention was made, in part, with government support under grant(contract) nos. P30-CA 16672; 1RO1-CA109570; 1RO1-CA119127; PO1-CA099031project 4; and P50 CA116100 01 project 4 awarded by the NationalInstitutes of Health and grant no. DAMD17-02-1-0694 awarded by theDepartment of Defense. The government has certain rights to thisinvention.

1. FIELD OF THE INVENTION

This application relates to combination therapies including triciribinecompounds and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, and compositions withreduced toxicity for the treatment and prevention of tumors, cancer, andother disorders associated with abnormal cell proliferation.

2. BACKGROUND OF THE INVENTION

Cancer is an abnormal growth of cells. Cancer cells rapidly reproducedespite restriction of space, nutrients shared by other cells, orsignals sent from the body to stop reproduction. Cancer cells are oftenshaped differently from healthy cells, do not function properly, and canspread into many areas of the body. Abnormal growths of tissue, calledtumors, are clusters of cells that are capable of growing and dividinguncontrollably. Tumors can be benign (noncancerous) or malignant(cancerous). Benign tumors tend to grow slowly and do not spread.Malignant tumors can grow rapidly, invade and destroy nearby normaltissues, and spread throughout the body.

Cancers are classified according to the kind of fluid or tissue fromwhich they originate, or according to the location in the body wherethey first developed. In addition, some cancers are of mixed types.Cancers can be grouped into five broad categories, carcinomas, sarcomas,lymphomas, leukemias, and myelomas, which indicate the tissue and bloodclassifications of the cancer. Carcinomas are cancers found in bodytissue known as epithelial tissue that covers or lines surfaces oforgans, glands, or body structures. For example, a cancer of the liningof the stomach is called a carcinoma. Many carcinomas affect organs orglands that are involved with secretion, such as breasts that producemilk. Carcinomas account for approximately eighty to ninety percent ofall cancer cases. Sarcomas are malignant tumors growing from connectivetissues, such as cartilage, fat, muscle, tendons, and bones. The mostcommon sarcoma, a tumor on the bone, usually occurs in young adults.Examples of sarcoma include osteosarcoma (bone) and chondrosarcoma(cartilage). Lymphoma refers to a cancer that originates in the nodes orglands of the lymphatic system, whose job it is to produce white bloodcells and clean body fluids, or in organs such as the brain and breast.Lymphomas are classified into two categories: Hodgkin's lymphoma andnon-Hodgkin's lymphoma. Leukemia, also known as blood cancer, is acancer of the bone marrow that keeps the marrow from producing normalred and white blood cells and platelets. White blood cells are needed toresist infection. Red blood cells are needed to prevent anemia.Platelets keep the body from easily bruising and bleeding. Examples ofleukemia include acute myelogenous leukemia, chronic myelogenousleukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.The terms myelogenous and lymphocytic indicate the type of cells thatare involved. Finally, myelomas grow in the plasma cells of bone marrow.In some cases, the myeloma cells collect in one bone and form a singletumor, called a plasmacytoma. However, in other cases, the myeloma cellscollect in many bones, forming many bone tumors. This is called multiplemyeloma.

Tumor induction and progression are often the result of accumulatedchanges in the tumor-cell genome. Such changes can include inactivationof cell growth inhibiting genes, or tumor suppressor genes, as well asactivation of cell growth promoting genes, or oncogenes. Hundreds ofactivated cellular oncogenes have been identified to date in animalmodels, however, only a small minority of these genes have proven to berelevant to human cancers (Weinberg et al., Oncogenes and the MolecularOrigins of Cancer, 1989. Cold Spring Harbor, N.Y.; Stanbridge J. et al.,Cell, 1990. 63: p 867-874; Godwin et al., in Gynecological oncology:principles and practice, Hoskins W. J., Perez C. A. and Young R. C.(eds.), 1992. pp 87-116, Lippincott, Philadelphia). The activation ofoncogenes in human cancers can result from factors such as increasedgene copy number or structural changes. These factors can cause numerouscellular effects, for example, they can result in overexpression of agene product. Several oncogenes involved in human cancer can beactivated through gene overexpression.

It has become apparent that the successive genetic aberrations acquiredby cancer cells result in defects in regulatory signal transductioncircuits that govern normal cell proliferation, differentiation andprogrammed cell death (Hanahan, D. and Weinberg R. A., Cell, 2000.100(1): p. 57-700). This in turn results in fundamental defects in cellphysiology which dictate malignancy. These defects include: a) selfsufficiency in growth signals (i.e. overexpression of growth factorreceptor tyrosine kinases such as EGFR and aberrant activation ofdownstream signal transduction pathways such as Ras/Raf/Mek/Erk 1/2 andRas/PI3K/Akt), b) resistance to anti-growth signals (i.e. lowerexpression of TGFβ and its receptor), c) evading apoptosis (i.e. loss ofproapoptotic p53; overexpression of pro-survival Bcl-2; hyperactivationof survival pathways such as those mediated by PI3K/Akt), d) sustainedangiogenesis (i.e. high levels of secretion of VEGF) and f) tissueinvasion and metastasis (i.e. extracellular proteases and prometastaticintegrins) (Hanahan, D. and Weinberg R. A., Cell, 2000. 100(1): p.57-700).

Receptor tyrosine kinases such as EGFR, ErbB2, VEGFR and insulin-likegrowth factor I receptor (IGF-1R) are intimately involved in thedevelopment of many human cancers including colorectal pancreatic,breast and ovarian cancers (Khaleghpour K. et al., Carcinogenesis, 2004.25(2): p. 241-8.; Sekharam M. et al., Cancer Res, 2003. 63(22): p.7708-16). Binding of ligands such as EGF, VEGF and IGF-1 to theirreceptors promotes stimulation of the intrinsic tyrosine kinaseactivity, autophosphorylation of specific tyrosines in the cytoplasmicdomain of the receptors and recruitment of signaling proteins thattrigger a variety of complex signal transduction pathways (Olayioye M.A. et al., Embo J, 2000. 19(13): p. 3159-67; Porter A. C. andVaillancourt R. R., Oncogene, 1998. 17(11 Reviews): p. 1343-52). This inturn leads to the activation of many tumor survival and oncogenicpathways such as the Ras/Raf/Mek/Erk ½, JAK/STAT3 and PI3K/Akt pathways.Although all three pathways have been implicated in colon, pancreatic,breast and ovarian oncogenesis, those that are mediated by Akt have beenshown to be critical in many steps of malignant transformation includingcell proliferation, anti-apoptosis/survival, invasion and metastasis andangiogenesis (Datta S. R. et al., Genes Dev, 1999. 13(22): p. 2905-27).

Akt is a serine/threonine protein kinase (also known as PKB), which has3 family members Akt1, Akt2 and Akt3. Stimulation of cells with growthor survival factors results in recruitment to the receptors of the lipidkinase phosphoinositide-3-OH-kinase (PI3K) which phosphorylatesphosphoinositol-4,5-biphosphate (PIP₂) to PIP₃ which recruits Akt to theplasma membrane where it can be activated by phosphorylation on Thr308and Ser473 (Akt1), Thr308 and Ser474 (Akt2) and Thr308 and Ser472 (Akt3)(Datta S. R. et al., Genes Dev, 1999. 13(22): p. 2905-27). Thus, PI3Kactivates Akt by phosphorylating PIP2 and converting to PIP3. Thephosphatase PTEN dephosphorylates PIP3 to PIP2 and hence prevents theactivation of Akt.

The majority of human cancers contain hyperactivated Akt (Datta S. R. etal., Genes Dev, 1999. 13(22): p. 2905-27; Bellacosa A. et al., Int JCancer, 1995. 64(4): p. 280-5; Sun M. et al., Am J Pathol, 2001. 159(2):p. 431-7). In particular, Akt is overexpressed and/or hyperactivated in57%, 32%, 27% and 36% of human colorectal, pancreatic, breast andovarian cancers, respective) (Roy H. K. et al., Carcinogenesis, 2002.23(1): p. 201-5; Altomare D. A. et al., J Cell Biochem, 2003. 88(1): p.470-6; Sun M. et al., Cancer Res, 2001. 61(16): p. 5985-91; Stal O. etal., Breast Cancer Res, 2003. 5(2): p. R37-44; Cheng J. Q. et al., ProcNatl Acad Sci USA, 1992. 89(19): p. 9267-71; Yuan Z. Q. et al.,Oncogene, 2000. 19(19): p. 2324-30). Hyperactivation of Akt is due toamplification and/or overexpression of Akt itself as well as geneticalterations upstream of Akt including overexpression of receptortyrosine kinases and/or their ligands and deletion of the phosphatasePTEN (Khaleghpour K. et al., Carcinogenesis, 2004. 25(2): p. 241-8;Sekharam M. et al., Cancer Res, 2003. 63(22): p. 7708-16; Cohen B. D. etal., Biochem Soc Symp, 1998. 63: p. 199-210; Muller W. J. et al.,Biochem Soc Symp, 1998. 63: p. 149-57; Miller W. E. et al., J Virol,1995. 69(7): p. 4390-8; Slamon D. J. et al., Science, 1987. 235(4785):p. 177-82; Andrulis I. L. et al., J Clin Oncol, 1998. 16(4): p. 1340-9).Proof-of-concept of the involvement of Akt in oncogenesis has beendemonstrated preclinically by showing that ectopic expression of Aktinduces malignant transformation and promotes cell survival (Sun M. etal., Am J Pathol, 2001. 159(2): p. 431-7; Cheng J. Q. et al., Oncogene,1997. 14(23): p. 2793-801) and that disruption of Akt pathways inhibitscell growth and induces apoptosis (Jetzt A. et al. Cancer Res, 2003.63(20): p. 6697-706).

Current treatments of cancer and related diseases have limitedeffectiveness and numerous serious unintended side effects. Despitedemonstrated clinical efficacy of many anti-cancer drugs, severesystemic toxicity often halts the clinical development of promisingchemotherapeutic agents. Further, overexpression of receptor tyrosinekinases such as EGFR and their ligands such as IGF-1, Akt overexpressionand/or loss of PTEN (all of which result in hyperactivation of Akt) areassociated with poor prognosis, resistance to chemotherapy and shortenedsurvival time of cancer patients. Current research strategies emphasizethe search for effective therapeutic modes with less risk.

Thus, a combination of a triciribine compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab, holdspromise as a potential combination therapy for treating tumors, cancer,and abnormal cell proliferation while synergistically reducing toxicityor adverse side effects caused by currently administered cancercompounds.

3. SUMMARY OF THE INVENTION

The present invention provides novel therapeutic regimens oftriciribine, triciribine phosphate and related compounds in combinationwith a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, to treat tumors or cancer in asubject while limiting systemic toxicity. The invention is based on thediscovery that tumors or cancers, which overexpress Akt kinase areparticularly sensitive to the cytotoxic effects of TCN and relatedcompounds and a synergistic affect would arise with a combination of amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof. The inventors have determined, contraryto the prior art and experience, how to successfully use triciribine anda molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, to treat tumors and cancer by one or acombination of (i) administering triciribine and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, to patients who exhibit enhanced sensitivity to thetriciribine compound and or the trastuzumab or a salt thereof; (ii) useof a described dosage level that minimizes the toxicity of thetriciribine compound and/or the a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, but yetstill exhibits efficacy; or (iii) use of a described dosage regimen thatminimizes the toxicity of the triciribine compound and/or the a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof.

In one aspect of the present invention, the invention encompasses acomposition including:

(i) a compound of the formula I:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate;

wherein R^(x) and R^(y) are independently hydrogen, optionallysubstituted phosphate; acyl (including lower acyl); amide, alkyl(including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl;

(ii) a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof; and

(iii) a pharmaceutically acceptable carrier.

In another embodiment, the invention encompasses a method of treating atumor or cancer in a mammal including administering to the mammal aneffective amount of a composition including:

(i) a compound of formula I:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate;

wherein R^(x) and R^(y) are independently hydrogen, optionallysubstituted phosphate; acyl (including lower acyl); amide, alkyl(including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl; and

(ii) a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof.

Methods are useful to treat tumors and cancers that are particularlysusceptible to the toxic effects of TCN, TCN-P and/or related compounds.In another embodiment, methods are provided for treating a tumor in amammal, particularly a human that includes (i) obtaining a biologicalsample from the tumor; (ii) determining whether the tumor overexpressesan Akt kinase, and (iii) treating the tumor that overexpresses Aktkinase with triciribine, triciribine phosphate or a related compound incombination with trastuzumab or a salt thereof, as described herein. Inanother embodiment, the level of Akt kinase expression can be determinedby assaying the tumor or cancer for the presence of a phosphorylated Aktkinase, for example, by using an antibody that can detect thephosphorylated form. In another embodiment, the level of Akt expressioncan be determined by assaying a tumor or cancer cells obtained from asubject and comparing the levels to a control tissue. In certainembodiments, the Akt can be overexpressed at least 2, 2.5, 3 or 5 foldin the cancer sample compared to the control. In certain embodiments,the overexpressed Akt kinase can be a hyperactivated and phosphorylatedAkt kinase.

In some aspects of the present invention triciribine, triciribinephosphate or a related compound increases and/or restores sensitivity toa molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof. In some embodiments, triciribine,triciribine phosphate or a related compound compensates for PTENdeficiency. In some embodiments of the present invention, triciribine,triciribine phosphate or a related compound in combination with amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, works synergistically. In otherembodiments, triciribine, triciribine phosphate or a related compound incombination with a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, induces apoptosis.In other embodiments, triciribine, triciribine phosphate or a relatedcompound in combination with a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, decreasesactivity of PI3K.

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TCN and related compounds. Inanother embodiment, such dosing regimens minimize or eliminate toxicside effects, including, but not limited to, hepatoxicity,thrombocytopenia, hyperglycemia, vomiting, hypocalcemia, anemia,hypoalbunemia, myelosuppression, hypertriglyceridemia, hyperamylasemia,diarrhea, stomachitis and/or fever. In another embodiment, theadministration of TCN, TCN-P or related compounds provides at least apartial, such as at least 15, 20 or 30%, or complete response in vivo inat least 15, 20, or 25% of the subjects.

In another embodiment, a method is provided to treat a subject who hasbeen diagnosed with a tumor by administering to the subject an effectiveamount of TCN, TCN-P or a related compound and a molecule that modulatesthe HER2/neu (erbB2) receptor, for example, trastuzumab or a saltthereof, according to a dosing schedule that includes administering thetriciribine compound and the a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof,approximately one time per week for approximately three weeks followedby a one week period wherein the triciribine compound and the moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, is not administered. In another embodiment, methodsare provided to treat tumor or cancer in a subject by administering tothe subject a dosing regimen of 10 mg/m² or less of TCN, TCN-P or arelated compound and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, each one time perweek. In another embodiment, the triciribine compound and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, can be administered as a single bolus dose over ashort period of time, for example, about 5, 10 or 15 minutes. In furtherembodiments, dosing schedules are provided in which the triciribinecompound and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, are administered viacontinuous infusion for at least 24, 48, 72, 96, or 120 hours. Incertain embodiments, the continuous administration can be repeated atleast once a week, once every two weeks and/or once a month. In otherembodiments, the triciribine compound and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,can be administered at least once every three weeks. In furtherembodiments, the compounds can be administered at least once a day forat least 2, 3, 4 or 5 days.

In further embodiments, the triciribine compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, as disclosed herein can be administered to patients in anamount that is effective in causing tumor regression. The administrationthe triciribine compound and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, canprovide at least a partial, such as at least 15, 20 or 30%, or completeresponse in vivo in at least 15-20% of the subjects. In certainembodiments, at least 2, 5, 10, 15, 20, 30 or 50 mg/m² of thetriciribine compound and at least about 20, 50, 100, 150, 200, 300, 400,600, 800, or 1000 mg a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof or a salt thereof,disclosed herein can be administered to a subject. The administration ofthe triciribine compound and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beconducted according to any of the therapeutic regimens disclosed herein.In particular embodiments, the dosing regimen can include administeringless than 20 mg/m² of the triciribine compound and less than 400 mg oftrastuzumab or a salt thereof. In one embodiment, less than 10 mg/m² ofthe triciribine compound and less than 400 mg trastuzumab or a saltthereof or a salt thereof can be administered once a week. In furtherembodiments, dosages of or less than 2 mg/m², 5 mg/m², 10 mg/m², and/or15 mg/m² of the triciribine compound and less than 400 mg, 300 mg, 200mg, 100 mg, and/or 50 mg of a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beadministered to a subject. In another embodiment, less than 10 mg/m² canbe administered to a subject via continuous infusion for at least fivedays. In particular embodiments, the triciribine compound and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, can be used for the treatment of pancreatic,prostate, colo-rectal and/or ovarian cancer.

In another embodiment, the triciribine compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, and/or can be used in a therapeutic regimen to preventand/or treat a carcinoma, sarcoma, lymphoma, leukemia, and/or myeloma.In other embodiments of the invention, the triciribine compound and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, can be used to treat solid tumors. Instill further embodiments, the triciribine compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, and compositions disclosed herein can be used for thetreatment of a tumor or cancer, such as, but not limited to cancer ofthe following organs or tissues: breast, prostate, bone, lung, colon,including, but not limited to colorectal, urinary, bladder, non-Hodgkinlymphoma, melanoma, kidney, renal, pancreas, pharnx, thyroid, stomach,brain, and/or ovaries. In a particular embodiment, the triciribinecompound and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, can be used for thetreatment of pancreatic, breast, colorectal and/or ovarian cancer. Infurther embodiments of the present invention, the triciribine compoundand a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, disclosed herein can be used inthe treatment of angiogenesis-related diseases. In certain embodiments,methods are provided to treat leukemia via continuous infusion of thetriciribine compound and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, via continuousinfusion for at least 24, 48, 72 or 96 hours. In other embodiments, thecontinuous infusion can be repeated, for example, at least once everytwo, three or four weeks.

In a particular embodiment, there is provided a method for the treatmentof tumors, cancer, and others disorders associated with an abnormal cellproliferation in a host, the method includes administering to the hostan effect amount of the triciribine compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, optionally in combination with a pharmaceuticallyacceptable carrier.

In one aspect, the triciribine compound and a molecule that modulatesthe HER2/neu (erbB2) receptor, for example, trastuzumab or a saltthereof, and compositions can be administered in combination and canform part of the same composition, or be provided as a separatecomposition for administration at the same time or a different time.

In other embodiments, the triciribine compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, as disclosed herein can be used to treat tumors or cancersresistant to one or more known anti-cancer drugs, including theembodiments of tumors or cancers and compounds disclosed herein. In oneembodiment, the triciribine compound and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,as disclosed herein is administered in an effective amount for thetreatment of a patient with a drug resistant tumor or cancer, forexample, multidrug resistant tumors or cancer including, but not limitedto, those resistant to taxol alone, rapamycin, tamoxifen, cisplatin,and/or gefitinib (iressa).

In certain embodiments, a method is provided including administering toa host in need thereof an effective amount of a triciribine compound anda molecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, disclosed herein, or pharmaceuticalcomposition including a triciribine compound and trastuzumab or a saltthereof, in an effective amount for the treatment of the treatment oftumors, cancer, and others disorders associated with an abnormal cellproliferation in a host.

In another embodiment, a method for the treatment of a tumor or canceris provided including an effective amount of a triciribine compounddisclosed herein, or a salt, isomer, prodrug or ester thereof and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, to an individual in need thereof, whereinthe cancer is for example, carcinoma, sarcoma, lymphoma, leukemia, ormyeloma. The compound, or salt, isomer, prodrug or ester thereof, isoptionally provided in a pharmaceutically acceptable compositionincluding the appropriate carriers, such as water, which is formulatedfor the desired route of administration to an individual in needthereof. Optionally the compound is administered in combination oralternation with at least one additional therapeutic agent for thetreatment of tumors or cancer.

Also within the scope of the invention is the use of a compounddisclosed herein or a salt, prodrug or ester thereof in the treatment ofa tumor or cancer, optionally in a pharmaceutically acceptable carrier;and the use of a triciribine compound and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,disclosed herein or a salt, prodrug or ester thereof in the manufactureof a medicament for the treatment of cancer or tumor, optionally in apharmaceutically acceptable carrier.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1D demonstrates the identification of API-2 (triciribine) as acandidate of Akt inhibitor from the NCI Diversity Set. FIG. 1Aillustrates the chemical structure of API-2 (triciribine). FIG. 1Bdemonstrates that API-2 inhibits phosphorylation levels of AKT2 inAKT2-transformed NIH3T3 cells. Wile type AKT2-transformed NIH3T3 cellswere treated with API-2 (1 μM) for indicated times and subjected toimmunoblotting analysis with anti-phospho-Akt-T308 and -S473 antibodies(top and middle panels). The bottom panel shows expression of totalAKT2. In FIG. 1C, it is shown that API-2 inhibits three isoforms of Akt.HEK293 cells were transfected with HA-Akt1, -AKT2 and -AKT3 and treatedwith API-2 (1 μM) or wortmannin (15 μM) prior to EGF stimulation, thecells were lysed and immunoprecipitated with anti-HA antibody. Theimmunoprecipitates were subjected to in vitro kinase assay (top) andimmunoblotting analysis with anti-phospho-Akt-T308 (bottom) antibody.Middle panel shows expression of transfected Akt1, AKT2 and AKT3. FIG.1D illustrates that API-2 did not inhibit Akt in vitro. In vitro kinaseassay of constitutively active AKT2 recombinant protein in a kinasebuffer containing 1 μM API-2 (lane 3).

FIG. 2A-2F demonstrates that API-2 does not inhibit PI3K, PDK1 and theclosely related members of AGC kinase family. FIG. 2A demonstrates an invitro PI3K kinase assay. HEK293 cells were serum-starved and treatedwith API-2 (1 μM) or Wortmannin (15 μM) fro 30 minutes prior to EGFstimulation. Cells were lysed and immunoprecipitated with anti-p110antibody. The immunoprecipitates were subjected to in vitro kinase assayusing PI-4-P as substrate. FIG. 2B illustrates the effect of API-2 on invitro PDK1 activation (top panel), closed circles show inhibition byAPI-2. Open circles show inhibition by the positive controlstaurosporine, which is a potent PDK1 inhibitor (IC50=5 nM). Bottompanels are immunoblotting analysis of HEK293 cells that were transfectedwith Myc-PDK1 and treated with wortmannin or API-2 prior to EGFstimulation. The immunoblots were detected with indicated antibodies.FIG. 2C illustrates an immunoblot analysis of phosphorylation levels ofPKC with anti-phospho-PKC T638 (top) and total PKC (bottom) antibodiesfollowing treatment with API-2 or a nonselective PKC inhibitorRo31-8220. FIG. 2D shows an in vitro SGK kinase assay. HEK293 cells weretransfected with HA-SGK and treated with API-2 or wortmannin prior toEGF stimulation. In vitro kinase was performed with HA-SGKimmunoprecipitates using MBP as substrate (top). Bottom panel shows theexpression of transfected HA-SGK. FIG. 2E illustrates the results of aPKA kinase assay. Immuno-purified PKA was incubated in ADB buffer(Upstate Biotechnology Inc) containing indicated inhibitors (API-2 orPKAI) and substrate Kemptide. The kinase activity was quantified. InFIG. 2F, a western blot is shown. OVCAR3 cells were treated with API-2for indicated times. Cell lysates were immunoblotted with indicatedanti-phospho-antibodies (panels 1-4) and anti-actin antibody (bottom).

FIG. 3A-3C-7 demonstrates that API-2 inhibits Akt activity and cellgrowth and induces apoptosis in human cancer cells with elevated Akt.FIG. 3A is a western blot, following treatment with API-2,phosphorylation levels of Akt were detected with anti-phospho-Akt-T308antibody in indicated human cancer cell lines. The blots were reprobedwith anti-total Akt antibody (bottom panels). In FIG. 3B, a cellproliferation assay is shown. Cell lines as indicated in the figure weretreated with different doses of API-2 for 24 h and 48 h and thenanalyzed with CellTiter 96 Cell Proliferation Assay kit (Promega). FIG.3C provides an apoptosis analysis. Cells were treated with API-2 andstained with annexin V and PI and analyzed by FACScan.

FIG. 4A-4E shows that API-2 inhibits downstream targets of Akt andexhibits anti-tumor activity in cancer cell lines with elevated Akt inmouse xenograft. In FIG. 4A, it is demonstrated that API-2 inhibits Aktphosphorylation of tuberin, Bad, AFX and GSK-3. Following treatment withAPI-2, OVAR3 cells were lysed and immunoblotted with indicatedantibodies. FIG. 4B shows that API-2 inhibits tumor growth. Tumor cellswere subcutaneously injected into nude mice with low level of Akt cellson left side and elevated level of Akt cells on right side. When thetumors reached an average size of about 100-150 mm³, animals weretreated with either vehicle or 1 mg/kg/day API-2. Each measurementrepresents an average of 10 tumors. FIG. 4C illustrates a representationof the mice with OVCAR3 (right) and OVCAR5 (left) xenograft treated withAPI-2 or vehicle (control). FIG. 4D shows examples of tumor size(bottom) and weight (top) at the end of experiment. In FIG. 4E,immunoblot analysis of tumor lysates was performed withanti-phospho-Akt-5473 (top) and anti-AKT2 (bottom) antibodies inOVCAR-3-derived tumors that were treated (T3 and T4) and untreated (T1and T2) with API-2.

FIG. 5 shows that API-2 (triciribine) inhibits Akt kinase activity invitro. In vitro kinase assay was performed with recombinant of PDK1 andAkt in a kinase buffer containing phosphatidylinositol-3,4,5-P3 (PIP3),API-2 and histone H2B as substrate. After incubation of 30 min, thereactions were separated by SDS-PAGE and exposed in a film.

FIG. 6A-6D provides the mRNA and amino acid sequence of human Akt1,restriction enzyme sites are also noted.

FIG. 7A-7D provides the mRNA and amino acid sequence of human Akt2restriction enzyme sites are also noted.

FIG. 8A-8D provides the mRNA and amino acid sequence of human Akt3restriction enzyme sites are also noted.

FIG. 9A-9C shows growth inhibition by the combination of trastuzumab andAkt/mTOR pathway inhibitors. PTEN anti-sense or non-specificoligonucleotide transfected BT474.m1 cells were treated with inhibitorsof the Akt/mTOR pathway alone or in combination with trastuzumab and therelative cell growth was assessed. FIG. 9A shows a panel of Akt/mTORinhibitors. Growth inhibition was assessed in PTEN AS transfectedBT474.m1 cells. The doses shown are: Triciribine (TCN) 1 μM; RAD001 0.2nM; QLT0267 10 μM; KP 372-1 0.05 μM; 4ADPIB 5 μM; Edelfosine 7.5 μM; andtrastuzumab (Ttzm) 2 μg/ml. The standard deviation (SD) in the percentgrowth inhibition is indicated. Results shown are the combined data from2-3 experiments with triplicates of each treatment within eachexperiment. FIG. 9B shows TCN inhibits cell growth in combination withtrastuzumab. BT474.m1 cells were transfected with PTEN ASoligonucleotide or non-specific (NS) oligonucleotide, treated withtrastuzumab and TCN, alone and in combination, at multiple doses of TCNand assayed for growth inhibition. Trastuzumab was administered at asingle concentration. FIG. 7C shows RAD001 inhibits cell growth incombination with trastuzumab. BT474.m1 cells were transfected with PTENAS oligonucleotide or non-specific (NS) oligonucleotide, treated withtrastuzumab and RAD001, alone and in combination, at multiple doses ofRAD001 and assayed for growth inhibition. For FIGS. 9B and 9C: *indicates a significant difference in growth inhibition followingcombination treatment as compared to either trastuzumab or TCN/TAD001alone. P<0.05 was considered significant. Error bars depict the SEM.

FIG. 10 shows the synergistic effects on apoptosis. Twenty-four hoursafter plating, PTEN AS and NS transfected BT474.m1 cells were treated asindicated with trastuzumab (Ttzm), TCN and/or RAD001 at the followingconcentrations: trastuzumab 2 mg/ml; triciribine 2.5 μM; RAD001 0.4 nM.Apo-BrdU Tunel assays were performed to assess apoptosis. The experimentwas performed 3 times and the data shown is the mean apoptosis.Errorbars depitc the standard deviation. Trastuzumab+triciribine treatmentsignificantly induced apoptosis (p<0.01) as compared with all othertreatments.

FIG. 11A-11B shows inhibition of Akt and p70S6K activity. To evaluatethe effects of these drugs on the Akt/mTOR pathway, PTEN AS and NSoligonucleotides were transfected int BT474.m1 cells. Two days later,the cells were treated for 2 hours with trastuzumab and triciribine(TCN) (FIG. 11A) or trastuzumab and RAD001 (FIG. 11B). Total celllysates were collected, separated by SDS-PAGE and immunoblotted asindicated. The concentration of trastuzumab was 2 μg/ml, triciribine was2.5 μM and RAD001 was 0.4 nM. The experiments were repeated at leasttwice to insure that results were reproducible.

FIG. 12A-12B shows combination treatments inhibited tumor growth in aSCID mice xenograft model. SCID mice received BT474.m1 breast cancercell xenografts in mammary fat pad. The xenografts grew for 3 weeks togenerate tumors with an average size of 100-150 mm³. PTEN antisenseoligonucleotides, trastuzumab, triciribine (FIG. 12A) and RAD001 (FIG.12B) were administered. The tumors were measured twice weekly withcalipers and tumor size was averaged for each treatment group. Errorbars denote the standard error of the mean. * indicates a significantdifference in growth inhibition following combination treatment ascompared to either trastuzumab (Ttzm), TCN or DMSO alone. P<0.05 wasconsidered significant.

5. DETAILED DESCRIPTION OF THE INVENTION

The inventors have determined, contrary to the prior art and experience,how to successfully use triciribine compounds in combination with amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, to treat tumors and cancer by one or acombination of (i) administering triciribine and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, only to patients, who according to a diagnostic testdescribed below, exhibit enhanced sensitivity to the triciribinecompound and/or the trastuzumab or a salt thereof; (ii) using adescribed dosage level that minimizes the toxicity of the triciribinecompound and/or the trastuzumab or a salt thereof but yet still exhibitsefficacy; or (iii) using a described dosage regimen that minimizes thetoxicity of the triciribine compound and/or the trastuzumab or a saltthereof

5.1. DEFINITIONS

As used herein, the term “compounds of the invention” refers tocompounds of formula I, compounds of formula II, and combinationsthereof.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth, i.e., proliferative disorders. Examples ofsuch proliferative disorders include cancers such as carcinoma,lymphoma, blastoma, sarcoma, and leukemia, as well as other cancersdisclosed herein. More particular examples of such cancers includebreast cancer, prostate cancer, colon cancer, squamous cell cancer,small-cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, cervical cancer, ovarian cancer, livercancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,endometrial carcinoma, kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); pancreatic cancer; retinoblastoma; rhabdomyosarcoma;rectal cancer; cancer of the respiratory system; sarcoma; skin cancer;stomach cancer; testicular cancer; uterine cancer; cancer of the urinarysystem, as well as other carcinomas and sarcomas.

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography), or by needle aspirations. The use of these lattertechniques is more common in early detection. Molecular and phenotypicanalysis of cancer cells within a tissue will usually confirm if thecancer is endogenous to the tissue or if the lesion is due to metastasisfrom another site.

The term alkyl, as used herein, unless otherwise specified, includes asaturated straight, branched, or cyclic, primary, secondary, or tertiaryhydrocarbon of for example C₁ to C₂₄, and specifically includes methyl,trifluoromethyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl,t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl, hexyl, isohexyl,cyclohexyl, cyclohexylmethyl, 3-methylpentyl, 2,2-dimethylbutyl, and2,3-dimethylbutyl. The alkyl is optionally substituted, e.g., with oneor more substituents such as halo (F, Cl, Br or I), (e.g. CF₃,2-Br-ethyl, CH₂F, CH₂Cl, CH₂CF₃ or CF₂CF₃), hydroxyl (e.g. CH₂OH), amino(e.g. CH₂NH₂, CH₂NHCH₃ or CH₂N(CH₃)₂), alkylamino, arylamino, alkoxy,aryloxy, nitro, azido (e.g. CH₂N₃), cyano (e.g. CH₂CN), sulfonic acid,sulfate, phosphonic acid, phosphate, or phosphonate, either unprotected,or protected as necessary, as known to those skilled in the art, forexample, as taught in Greene et al., Protective Groups in OrganicSynthesis, 1991, 2d Ed., John Wiley and Sons, NY, hereby incorporated byreference.

The term lower alkyl, as used herein, and unless otherwise specified,refers to a C₁ to C₄ saturated straight, branched, or if appropriate, acyclic (for example, cyclopropyl)alkyl group, including both substitutedand unsubstituted forms.

The term alkylamino or arylamino includes an amino group that has one ortwo alkyl or aryl substituents, respectively.

The term amino acid includes naturally occurring and synthetic α, β, γor δ amino acids, and includes but is not limited to, amino acids foundin proteins, i.e. glycine, alanine, valine, leucine, isoleucine,methionine, phenylalanine, tryptophan, proline, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartate, glutamate, lysine,arginine and histidine. In a preferred embodiment, the amino acid is inthe L-configuration. Alternatively, the amino acid can be a derivativeof alanyl, valinyl, leucinyl, isoleuccinyl, prolinyl, phenylalaninyl,tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl,tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl,argininyl, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleuccinyl,β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl,β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl,β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-argininyl orβ-histidinyl. When the term amino acid is used, it is considered to be aspecific and independent disclosure of each of the esters of a naturalor synthetic amino acid, including but not limited to α, β, γ or δglycine, alanine, valine, leucine, isoleucine, methionine,phenylalanine, tryptophan, proline, serine, threonine, cysteine,tyrosine, asparagine, glutamine, aspartate, glutamate, lysine, arginineand histidine in the D and L-configurations.

The term “protected” as used herein and unless otherwise definedincludes a group that is added to an oxygen, nitrogen, sulfur orphosphorus atom to prevent its further reaction or for other purposes. Awide variety of oxygen and nitrogen protecting groups are known to thoseskilled in the art of organic synthesis (see Greene and Wuts, ProtectiveGroups in Organic Synthesis, 1999, 3d Ed., John Wiley & Sons, Inc., NY).

The term aryl, as used herein, and unless otherwise specified, includesphenyl, biphenyl, or naphthyl, and preferably phenyl. The aryl group isoptionally substituted with one or more moieties such as halo, hydroxyl,amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonicacid, sulfate, phosphonic acid, phosphate, or phosphonate, eitherunprotected, or protected as necessary, as known to those skilled in theart, for example, as taught in Greene et al., Protective Groups inOrganic Synthesis, 1999, 3^(rd) Ed., John Wiley and Sons, NY.

The term alkaryl or alkylaryl includes an alkyl group with an arylsubstituent. The term aralkyl or arylalkyl includes an aryl group withan alkyl substituent.

The term halo, as used herein, includes chloro, bromo, iodo, and fluoro.

The term acyl includes a carboxylic acid ester in which the non-carbonylmoiety of the ester group is selected from straight, branched, or cyclicalkyl or lower alkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally comprise a phenyl group. The term “lower acyl” refers to anacyl group in which the non-carbonyl moiety is lower alkyl.

As used herein, the term “substantially free of” or “substantially inthe absence of” with respect to enantiomeric purity, refers to acomposition that includes at least 85% or 90% by weight, preferably 95%to 98% by weight, and even more preferably 99% to 100% by weight, of thedesignated enantiomer. In a preferred embodiment, in the methods andcompounds of this invention, the compounds are substantially free ofother enantiomers.

Similarly, the term “isolated” refers to a compound composition thatincludes at least 85% or 90% by weight, preferably 95% to 98% by weight,and even more preferably 99% to 100% by weight, of the compound, theremainder comprising other chemical species or enantiomers.

The term “independently” is used herein to indicate that the variable,which is independently applied, varies independently from application toapplication. Thus, in a compound such as R″XYR″, wherein R″ is“independently carbon or nitrogen,” both R″ can be carbon, both R″ canbe nitrogen, or one R″ can be carbon and the other R″ nitrogen.

The term “pharmaceutically acceptable salt or prodrug” is usedthroughout the specification to describe any pharmaceutically acceptableform (such as an ester, phosphate ester, salt of an ester or a relatedgroup) of a compound, which, upon administration to a patient, providesthe compound. Pharmaceutically acceptable salts include those derivedfrom pharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

The term “pharmaceutically acceptable esters” as used herein, unlessotherwise specified, includes those esters of one or more compounds,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of hosts without undue toxicity, irritation,allergic response and the like, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

The term “subject” as used herein refers to an animal, preferably amammal, most preferably a human. Mammals can include non-human mammals,including, but not limited to, pigs, sheep, goats, cows (bovine), deer,mules, horses, monkeys and other non-human primates, dogs, cats, rats,mice, rabbits or any other known or disclosed herein.

5.2. COMPOUNDS OF THE INVENTION

The present invention provides for the use of TCN, TCN-P and relatedcompounds in combination with a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, for use inparticular therapeutic regimens for the treatment of proliferativedisorders.

As used herein and unless otherwise indicated, the term “triciribinecompounds” and “triciribine and related compounds” refers to compoundshaving the following structures:

wherein each R2′, R3′ and R5′ are independently hydrogen, optionallysubstituted phosphate or phosphonate (including mono-, di-, ortriphosphate or a stabilized phosphate prodrug); acyl (including loweracyl); alkyl (including lower alkyl); amide, sulfonate ester includingalkyl or arylalkyl; sulfonyl, including methanesulfonyl and benzyl,wherein the phenyl group is optionally substituted with one or moresubstituents as for example as described in the definition of an arylgiven herein; optionally substituted arylsulfonyl; a lipid, including aphospholipid; an amino acid; a carbohydrate; a peptide; or cholesterol;or other pharmaceutically acceptable leaving group that, in vivo,provides a compound wherein R2′, R3′ or R5′ is independently H or mono-,di- or tri-phosphate;

wherein R^(x) and R^(y) are independently hydrogen, optionallysubstituted phosphate; acyl (including lower acyl); amide, alkyl(including lower alkyl); aromatic, polyoxyalkylene such aspolyethyleneglycol, optionally substituted arylsulfonyl; a lipid,including a phospholipid; an amino acid; a carbohydrate; a peptide; orcholesterol; or other pharmaceutically acceptable leaving group. In oneembodiment, the compound is administered as a 5′-phosphoether lipid or a5′-ether lipid.

R₁ and R₂ each are independently H, optionally substituted straightchained, branched or cyclic alkyl (including lower alkyl), alkenyl, oralkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl or heteroaryl,CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl, sulfonyl,alkylsulfonyl, arylsulfonyl, aralkylsulfonyl.

In one embodiment, R2′ and R3′ are hydrogen. In another embodiment, R2′and R5′ are hydrogen. In yet another embodiment, R2′, R3′ and R5′ arehydrogen. In yet another embodiment, R2′, R3′, R5′, R1 and R2 arehydrogen.

In another embodiment, the triciribine compound has the followingstructure:

wherein R₃ is H, optionally substituted straight chained, branched orcyclic alkyl (including lower alkyl), alkenyl, or alkynyl, NH₂, NHR⁴,N(R⁴)₂, aryl, alkoxyalkyl, aryloxyalkyl, or substituted aryl; and

each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl, or aryl. In a subembodiment, R₃ is a straight chainedC1-11 alkyl, iso-propyl, t-butyl, or phenyl.

In one embodiment, the triciribine compounds provided herein have thefollowing structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

wherein R₆ is H, alkyl, (including lower alkyl)alkenyl, alkynyl,alkoxyalkyl, hydroxyalkyl, arylalkyl, cycloalkyl, NH₂, NHR⁴, NR⁴R⁴, CF₃,CH₂OH, CH₂F, CH₂Cl, CH₂CF₃, C(Y³)₃, C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴,C(═O)-alkyl, C(═O)-aryl, C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴,C(═O)N(R⁴)₂, where each Y³ is independently H or halo; and

each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl, or aryl.

In a subembodiment, R₆ is ethyl, CH₂CH₂OH, or CH₂-phenyl.

In another embodiment, the triciribine compounds provided herein havethe following structure:

wherein R₇ is H, halo, alkyl (including lower alkyl), alkenyl, alkynyl,alkoxy, alkoxyalkyl, hydroxyalkyl, cycloalkyl, nitro, cyano, OH, OR⁴,NH₂, NHR⁴, NR⁴R⁴, SH, SR⁴, CF₃, CH₂OH, CH₂F, CH₂Cl, CH₂CF₃, C(Y³)₃,C(Y³)₂C(Y³)₃, C(═O)OH, C(═O)OR⁴, C(═O)-alkyl, C(═O)-aryl,C(═O)-alkoxyalkyl, C(═O)NH₂, C(═O)NHR⁴, C(═O)N(R⁴)₂, or N₃, where eachY³ is independently H or halo; and

each R⁴ independently is H, acyl including lower acyl, alkyl includinglower alkyl such as but not limited to methyl, ethyl, propyl andcyclopropyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxyalkyl,hydroxyalkyl.

In a subembodiment, R₇ is methyl, ethyl, phenyl, chloro or NH₂.

In another embodiment, the triciribine compounds provided herein havethe following structure:

In another embodiment, the triciribine compounds provided herein havethe following structure:

As used herein and unless otherwise indicated, the term “a molecule thatmodulates the HER2/neu (erbB2) receptor” refers to compounds which arerecombinant DNA-derived humanized monoclonal antibodies that selectivelybinds with high affinity in a cell-based assay to the extra-cellulardomain of the human epidermal growth factor receptor 2 protein, HER2. Incertain embodiments, the antibody is an IgG₁ kappa that contains humanframework regions with the complementary-determining regions of a murineantibody (4D5) that binds to HER2.

As used herein and unless otherwise indicated, the term “trastuzumab”refers to trastuzumab or a salt thereof. In certain embodiment, the termtrastuzumab refers to the compound with the DrugBank accession numberBTD00098 and the trade name Herceptin, which is a recombinant humanizedmonoclonal antibody directed against the human epidermal growth factorreceptor 2 (HER2). HER2 is overexpressed by many adenocarcinomas,particularly breast adenocarcinomas. Trastuzumab binds to HER2 on thecell surface of a tumor cell, thereby inducing an antibody-dependentcell-mediated cytotoxicity against tumor cells that overexpress HER2.

It is to be understood that the compounds disclosed herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is understood that the disclosure of acompound herein encompasses any racemic, optically active, polymorphic,or steroisomeric form, or mixtures thereof, which preferably possessesthe useful properties described herein, it being well known in the arthow to prepare optically active forms and how to determine activityusing the standard tests described herein, or using other similar testswhich are will known in the art. Examples of methods that can be used toobtain optical isomers of the compounds include the following:

(i) physical separation of crystals—a technique whereby macroscopiccrystals of the individual enantiomers are manually separated. Thistechnique can be used if crystals of the separate enantiomers exist,i.e., the material is a conglomerate, and the crystals are visuallydistinct;

(ii) simultaneous crystallization—a technique whereby the individualenantiomers are separately crystallized from a solution of the racemate,possible only if the latter is a conglomerate in the solid state;

(iii) enzymatic resolutions—a technique whereby partial or completeseparation of a racemate by virtue of differing rates of reaction forthe enantiomers with an enzyme

(iv) enzymatic asymmetric synthesis—a synthetic technique whereby atleast one step of the synthesis uses an enzymatic reaction to obtain anenantiomerically pure or enriched synthetic precursor of the desiredenantiomer;

(v) chemical asymmetric synthesis—a synthetic technique whereby thedesired enantiomer is synthesized from an achiral precursor underconditions that produce assymetry (i.e., chirality) in the product,which may be achieved using chiral catalysts or chiral auxiliaries;

(vi) diastereomer separations—a technique whereby a racemic compound isreacted with an enantiomerically pure reagent (the chiral auxiliary)that converts the individual enantiomers to diastereomers. The resultingdiastereomers are then separated by chromatography or crystallization byvirtue of their now more distinct structural differences and the chiralauxiliary later removed to obtain the desired enantiomer;

(vii) first- and second-order asymmetric transformations—a techniquewhereby diastereomers from the racemate equilibrate to yield apreponderance in solution of the diastereomer from the desiredenantiomer or where preferential crystallization of the diastereomerfrom the desired enantiomer perturbs the equilibrium such thateventually in principle all the material is converted to the crystallinediastereomer from the desired enantiomer. The desired enantiomer is thenreleased from the diastereomer;

(viii) kinetic resolutions—this technique refers to the achievement ofpartial or complete resolution of a racemate (or of a further resolutionof a partially resolved compound) by virtue of unequal reaction rates ofthe enantiomers with a chiral, non-racemic reagent or catalyst underkinetic conditions;

(ix) enantiospecific synthesis from non-racemic precursors—a synthetictechnique whereby the desired enantiomer is obtained from non-chiralstarting materials and where the stereochemical integrity is not or isonly minimally compromised over the course of the synthesis;

(x) chiral liquid chromatography—a technique whereby the enantiomers ofa racemate are separated in a liquid mobile phase by virtue of theirdiffering interactions with a stationary phase. The stationary phase canbe made of chiral material or the mobile phase can contain an additionalchiral material to provoke the differing interactions;

(xi) chiral gas chromatography—a technique whereby the racemate isvolatilized and enantiomers are separated by virtue of their differinginteractions in the gaseous mobile phase with a column containing afixed non-racemic chiral adsorbent phase;

(xii) extraction with chiral solvents—a technique whereby theenantiomers are separated by virtue of preferential dissolution of oneenantiomer into a particular chiral solvent;

(xiii) transport across chiral membranes—a technique whereby a racemateis placed in contact with a thin membrane barrier. The barrier typicallyseparates two miscible fluids, one containing the racemate, and adriving force such as concentration or pressure differential causespreferential transport across the membrane barrier. Separation occurs asa result of the non-racemic chiral nature of the membrane which allowsonly one enantiomer of the racemate to pass through.

In some embodiments, triciribine, triciribine phosphate (TCN-P),triciribine 5′-phosphate (TCN-P), or the DMF adduct of triciribine(TCN-DMF) are provided. TCN can be synthesized by any technique known toone skilled in the art, for example, as described in TetrahedronLetters, 1971. 49: p. 4757-4760. TCN-P can be prepared by any techniqueknown to one skilled in the art, for example, as described in U.S. Pat.No. 4,123,524. The synthesis of TCN-DMF is described, for example, inINSERM, 1978. 81: p. 37-82. Other compounds related to TCN as describedherein can be synthesized, for example, according to the methodsdisclosed in Gudmundsson K. S. et al., Nucleosides Nucleotides NucleicAcids, 2001. 20(10-11): p. 1823-1830; Porcari A. R. et al., J Med Chem,2000. 43(12): p. 2457-2463; Porcari A. R. et al., NucleosidesNucleotides, 1999. 18(11-12): p. 2475-2497; Porcari A. R. et al., J MedChem, 2000. 43(12): p. 2438-2448; Porcari A. R. et al., NucleosidesNucleotides Nucleic Acids, 2003. 22(12): p. 2171-2193; Porcari A. R. etal., Nucleosides Nucleotides Nucleic Acids, 2004. 23(1-2): p. 31-39;Schweinsberg P. D. et al., Biochem Pharmacol, 1981. 30(18): p.2521-2526; Smith K. L. et al., Bioorg Med Chem Lett, 2004. 14(13): p.3517-3520; Townsend L. B. et al., Nucleic Acids Symp Ser, 1986.1986(17): p. 41-44; and/or Wotring L. L. et al., Cancer Treat Rep, 1986.70(4): p. 491-7.

5.3. PHARMACEUTICALLY ACCEPTABLE SALTS AND PRODRUGS

In cases where compounds are sufficiently basic or acidic to form stablenontoxic acid or base salts, administration of the compound as apharmaceutically acceptable salt may be appropriate. Pharmaceuticallyacceptable salts include those derived from pharmaceutically acceptableinorganic or organic bases and acids. Suitable salts include thosederived from alkali metals such as potassium and sodium, alkaline earthmetals such as calcium and magnesium, among numerous other acids wellknown in the pharmaceutical art. In particular, examples ofpharmaceutically acceptable salts are organic acid addition salts formedwith acids, which form a physiological acceptable anion, for example,tosylate, methanesulfonate, acetate, citrate, malonate, tartarate,succinate, benzoate, ascorbate, α-ketoglutarate, and α-glycerophosphate.Suitable inorganic salts may also be formed, including, sulfate,nitrate, bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

Any of the nucleotides described herein can be administered as anucleotide prodrug to increase the activity, bioavailability, stabilityor otherwise alter the properties of the nucleoside. A number ofnucleotide prodrug ligands are known. In general, alkylation, acylationor other lipophilic modification of the mono, di or triphosphate of thenucleoside will increase the stability of the nucleotide. Examples ofsubstituent groups that can replace one or more hydrogens on thephosphate moiety are alkyl, aryl, steroids, carbohydrates, includingsugars, 1,2-diacylglycerol and alcohols. Many are described in R. Jonesand N. Bischofberger, Antiviral Research, 1995. 27: p. 1-17. Any ofthese can be used in combination with the disclosed nucleosides toachieve a desired effect.

In one embodiment, the triciribine or a related compound is provided as5′-hydroxyl lipophilic prodrug. Nonlimiting examples of U.S. patentsthat disclose suitable lipophilic substituents that can be covalentlyincorporated into the nucleoside, preferably at the 5′-OH position ofthe nucleoside or lipophilic preparations, include U.S. Pat. Nos.5,149,794, 5,194,654, 5,223,263, 5,256,641, 5,411,947, 5,463,092,5,543,389, 5,543,390, 5,543,391, and 5,554,728, all of which areincorporated herein by reference.

Foreign patent applications that disclose lipophilic substituents thatcan be attached to the triciribine or a related compound s of thepresent invention, or lipophilic preparations, include WO 89/02733, WO90/00555, WO 91/16920, WO 91/18914, WO 93/00910, WO 94/26273, WO/15132,EP 0 350 287, EP 93917054.4, and WO 91/19721.

Additional nonlimiting examples of derivatives of triciribine or relatedcompounds are those that contain substituents as described in thefollowing publications. These derivatized triciribine or relatedcompounds can be used for the indications described in the text orotherwise as antiviral agents, including as anti-HIV or anti-HBV agents.Ho D. H. W., Cancer Res., 1973 33: p. 2816-2820; Holy A. in Advances inAntiviral Drug Design, Vol. I, De Clercq (ed.), JAI Press, pp. 179-231;Hong C. I. et al. Biochem Biophys Rs Commun, 1979. 88: p. 1223-1229;Hong C. I. et al., J Med Chem, 1980. 28: p. 171-177; Hostetler K. Y. etal., J Biol Chem, 1990. 266: p. 11714-11717; Hostetler K. Y. et al,Antiviral Res, 1994. 24: p. 59-67; Hostetler K. Y. et al, AntimicrobialAgents Chemother, 1994. 38: p. 2792-2797; Hunston R. N. et al, J MedChem, 1984. 27: p. 440-444; Ji Y. H. et al, J Med Chem, 1990. 33: p.2264-2270; Jones A. S. et al., J Chem Soc Perkin Trans, 1984. I: p.1471-1474; Juodka B. A. and Smart J., Coll Czech Chem Comm, 1974. 39: p.363-968; Kataoka S. et al, Nucleic Acids Res Sym Ser, 1989. 21: p. 1-2;Kataoka S. et al, Heterocycles, 1991. 32: p. 1351-1356; Kinchington D.et al, Antiviral Chem Chemother, 1992. 3: p. 107-112; Kodama K. et al.,Jpn J Cancer Res, 1989. 80: p. 679-685; Korty M. and Engels J.,Naunyn-Schmiedeberg's Arch Pharmacol, 1979. 10: p. 103-111; Kumar A. etal., J Med Chem, 1990. 33: p. 2368-2375; LeBec C. and Huynh-dinh T.,Tetrahedron Lett, 1991. 32: p. 6553-6556; Lichtenstein J. et al., J BiolChem, 1960. 235: p. 457-465; Luethy J. et al., Mitt GegLebensmittelunters Hyg, 1981. 72: p. 131-133 (Chem. Abstr. 95, 127093);McGuigan C. et al., Nucleic Acids Res, 1989. 17: p. 6065-6075; McGuiganC. et al., Antiviral Chem Chemother, 1990. 1: p. 107-113; McGuigan C. etal, Antiviral Chem Chemother, 1990. 1: p. 355-360; McGuigan C. et al.,Antiviral Chem Chemother, 1990. 1: p. 25-33; McGuigan C. et al.,Antiviral Res, 1991. 15: p. 255-263; McGuigan C. et al., Antiviral Res,1992. 17: p. 311-321; McGuigan C. et al., Antiviral Chem Chemother,1993. 4: p. 97-101; McGuigan C. et al., J Med Chem, 1993. 36: p.1048-1052.

Alkyl hydrogen phosphonate derivatives of the anti-HIV agent AZT may beless toxic than the parent nucleoside analogue. Antiviral Chem.Chemother. 5: 271-277; Meyer R. B. et al., Tetrahedron Lett, 1973.269-272; Nagyvary J. et al., Biochem Biophys Res Commun, 1973. 55: p.1072-1077; Namane A. et al., J Med Chem, 1992. 35: p. 3939-3044; NargeotJ. et al., Natl. Acad. Sci. U.S.A., 1983. 80: p. 2395-2399; Nelson K. A.et al., J Am Chem Soc, 1987. 109: p. 4058-4064; Nerbonne J. M. et al.,Nature, 1984. 301: p. 74-76; Neumann J. M. et al., J Am Chem Soc, 11989. 111: p. 4270-4277; Ohno R. et al., Oncology, 1991. 48: p. 451-455.Palomino E. et al., J Med Chem, 1989. 32: p. 622-625; Perkins R. M. etal., Antiviral Res, 1993. 20(Suppl. I): p. 84; Piantadosi C. et al., JMed Chem, 1991. 34: 1408-1414; Pompon A. et al., Antiviral ChemChemother, 1994. 5: p. 91-98; Postemark T., Anu Rev Pharmacol, 1974. 14:p. 23-33; Prisbe E. J. et al., J Med Chem, 1986. 29: p. 671-675; PucchF. et al., Antiviral Res, 1993. 22: p. 155-174; Pugaeva V. P. et al.,Gig Trf Prof Zabol, 1969. 13: p. 47-48 (Chem. Abstr. 72, 212); Robins R.K., Pharm Res, 1984. 11-18; Rosowsky A. et al., J Med Chem, 1982. 25: p.171-178; Ross W., Biochem Pharm, 1961. 8: p. 235-240; Ryu E. K. et al.,J Med Chem, 1982. 25: p. 1322-1329; Saffhill R. and Hume W. J., ChemBiol Interact, 1986. 57: p. 347-355; Saneyoshi M. et al., Chem PharmBull, 1980. 28: p. 2915-2923; Sastry J. K. et al., Mol Pharmacol, 1992.41: p. 441-445; Shaw J. P. et al., 9th Annual AAPS Meeting, 1994. SanDiego, Calif. (Abstract). Shuto S. et al., Tetrahedron Lett, 1987. 28:p. 199-202; Shuto S. et al., Chem Pharm Bull, 1988. 36: p. 209-217. Onepreferred phosphate prodrug group is the S-acyl-2-thioethyl group, alsoreferred to as “SATE.”

Additional examples of prodrugs that can be used are those described inthe following patents and patent applications: U.S. Pat. Nos. 5,614,548,5,512,671, 5,770,584, 5,962,437, 5,223,263, 5,817,638, 6,252,060,6,448,392, 5,411,947, 5,744,592, 5,484,809, 5,827,831, 5,696,277,6,022,029, 5,780,617, 5,194,654, 5,463,092, 5,744,461, 4,444,766,4,562,179, 4,599,205, 4,493,832, 4,221,732, 5,116,992, 6,429,227,5,149,794, 5,703,063, 5,888,990, 4810,697, 5,512,671, 6,030,960,2004/0259845, 6,670,341, 2004/0161398, 2002/082242, 5,512,671,2002/0082242, and or PCT Publication Nos WO 90/11079, WO 96/39197,and/or WO 93/08807.

5.4. IN VIVO EFFICACY/DOSING REGIMENS

In another aspect of the present invention, dosing regimens are providedthat limit the toxic side effects of TCN and related compounds. In oneembodiment, such dosing regimens minimize the following toxic sideeffects, including, but not limited to, hepatoxicity, thrombocytopenia,hyperglycemia, vomiting, hypocalcemia, anemia, hypoalbunemia,myelosuppression, hypertriglyceridemia, hyperamylasemia, diarrhea,stomachitis and/or fever.

In another embodiment, the administration of TCN, TCN-P or relatedcompounds and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, provides at least a partialor complete response in vivo in at least 15-20% of the subjects. Inparticular embodiments, a partial response can be at least 15, 20, 25,30, 35, 40, 50, 55, 60, 65, 70, 75, 80 or 85% regression of the tumor.In other embodiments, this response can be evident in at least 15, 15,20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85 or 90% of thesubjects treated with the therapy. In further embodiments, such responserates can be obtained by any therapeutic regimen disclosed herein.

In other embodiments, methods are provided to treat a subject that hasbeen diagnosed with cancer by administering to the subject an effectiveamount of TCN, TCN-P or a related compound and a molecule that modulatesthe HER2/neu (erbB2) receptor, for example, trastuzumab or a saltthereof, according to a dosing schedule that includes administering thetriciribine compound and/or a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, one timeper week for three weeks followed by a one week period wherein the drugis not administered (i.e., via a 28 day cycle). In other embodiments,such 28 day cycles can be repeated at least 2, 3, 4, or 5 times or untilregression of the tumor is evident.

In further embodiments, a 42 day cycle is provided in which thecompounds disclosed herein can be administered once a week for fourweeks followed by a two week period in which the triciribine compoundand/or a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, is not administered. In otherembodiments, such 42 day cycles can be repeated at least 2, 3, 4, or 5times or until regression of the tumor is evident. In a particularembodiment, less than 12, less than 11 or less than 10 mg/m² of TCN,TCN-P or a related compound can be administered according to a 42 daycycle. In other particular embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15mg/m² of TCN, TCN-P or a related compound can be administered accordingto a 42 day cycle. In another particular embodiment, about 1 to about 50mg of a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, is administered. In a particularembodiment, 1, 5, 10, 15, 20, 25, 30, 35, or 40 mg of a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can be administered according to a 42 day cycle.

In another embodiment, methods are provided to treat cancer in a subjectby administering to the subject a dosing regimen of 10 mg/m² or less ofTCN, TCN-P or a related compound and less than about 30 mg of a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, one time per week. In particular embodiments, 0.5, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or10 mg/m² of TCN, TCN-P or a related compound as disclosed herein can beadministered one time per week In another particular embodiment, 1, 5,10, 15, 20, 25, 30, 35, or 40 mg of a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,can be administered one time per week.

In embodiments of the present invention, the compounds disclosed hereincan be administered simultaneously as a single bolus dose over a shortperiod of time, for example, about 5, 10, 15, 20, 30 or 60 minutes. Infurther embodiments, dosing schedules are provided in which thecompounds are administered simultaneously via continuous infusion for atleast 24, 48, 72, 96, or 120 hours. In certain embodiments, theadministration of the triciribine compound and/or a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, via continuous or bolus injections can be repeated at acertain frequency at least: once a week, once every two weeks, onceevery three weeks, once a month, once every five weeks, once every sixweeks, once every eight weeks, once every ten weeks and/or once everytwelve weeks. The type and frequency of administrations can be combinedin any manner disclosed herein to create a dosing cycle. The triciribinecompound and/or a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, can be repeatedlyadministered via a certain dosing cycles, for example as a bolusinjection once every two weeks for three months. The dosing cycles canbe administered for at least: one, two three, four five, six, seven,eight, nine, ten, eleven, twelve, eighteen or twenty four months.Alternatively, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15 or 20dosing cycles can be administered to a patient. The triciribine compoundand/or a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, can be administered according toany combination disclosed herein, for example, the triciribine compoundand/or a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, can be administered once a weekevery three weeks for 3 cycles.

In further embodiments, the compounds can be administered separately atleast once a day for at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 days. Suchadministration can be followed by corresponding periods in which thetriciribine compound and/or a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, are notadministered

The TCN, TCN-P and related compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,as disclosed herein can be administered to patients in an amount that iseffective in causing tumor regression. The administration of TCN, TCN-Por related compounds and trastuzumab or a salt thereof can provide atleast a partial, such as at least 15, 20 or 30%, or complete response invivo in at least 15-20% of the subjects. In certain embodiments, atleast 2, 5, 10, 15, 20, 30 or 50 mg/m² of a triciribine compounddisclosed herein can be administered to a subject. In certainembodiments, at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5,6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 12, 15, 17, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 165, 175, 200, 250,300, or 350 mg/m² of TCN, TCN-P or a related compound disclosed hereincan be administered to a subject. In certain embodiments, 1, 5, 10, 15,20, 25, 30, 35, or 40 mg of a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beadministered to a subject.

The administration of the compound can be conducted according to any ofthe therapeutic regimens disclosed herein. In particular embodiments,the dosing regimen includes administering less than about 20 mg/m² ofTCN and related compounds and less than about 30 mg of a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, either concurrently, sequentially, or conducted over aperiod of time. In one embodiment, less than 20 mg/m² of TCN or relatedcompounds can be administered once a week concurrently with less thanabout 30 mg of a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof. In another embodiment, lessthan 20 mg/m² of TCN or related compounds can be administered once aweek and less than about 30 mg of a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beadministered the following week.

In further embodiments, 2 mg/m², 5 mg/m², 10 mg/m², and/or 15 mg/m² ofTCN or a related compound and less than about 300, 250, 200, 150, or 100mg of a molecule that modulates the HER2/neu (erbB2) receptor, forexample, trastuzumab or a salt thereof, can be administered to asubject. In another embodiment, less than 10 mg/m² of a triciribinecompound and less than about 300 mg of a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,can be administered to a subject via continuous infusion for at leastfive days. The present invention provides for any combination of dosingtype, frequency, number of cycles and dosage amount disclosed herein.

5.5. Screening of Patient Populations

In another aspect of the present invention, methods are provided toidentify cancers or tumors that are susceptible to the toxic effects oftriciribine (TCN) and related compounds. In one embodiment, methods areprovided to treat a cancer or tumor in a mammal by (i) obtaining abiological sample from the tumor; (ii) determining whether the cancer ortumor overexpresses Akt kinase or hyperactivated and phosphorylated Aktkinase, and (iii) treating the cancer or tumor with triciribine or arelated compound as described herein. In one embodiment, the biologicalsample can be a biopsy. In other embodiments, the biological sample canbe fluid, cells and/or aspirates obtained from the tumor or cancer.

The biological sample can be obtained according to any technique knownto one skilled in the art. In one embodiment, a biopsy can be conductedto obtain the biological sample. A biopsy is a procedure performed toremove tissue or cells from the body for examination. Some biopsies canbe performed in a physician's office, while others need to be done in ahospital setting. In addition, some biopsies require use of ananesthetic to numb the area, while others do not require any sedation.In certain embodiments, an endoscopic biopsy can be performed. This typeof biopsy is performed through a fiberoptic endoscope (a long, thin tubethat has a close-focusing telescope on the end for viewing) through anatural body orifice (i.e., rectum) or a small incision (i.e.,arthroscopy). The endoscope is used to view the organ in question forabnormal or suspicious areas, in order to obtain a small amount oftissue for study. Endoscopic procedures are named for the organ or bodyarea to be visualized and/or treated. The physician can insert theendoscope into the gastrointestinal tract (alimentary tract endoscopy),bladder (cystoscopy), abdominal cavity (laparoscopy), joint cavity(arthroscopy), mid-portion of the chest (mediastinoscopy), or tracheaand bronchial system (laryngoscopy and bronchoscopy).

In another embodiment, a bone marrow biopsy can be performed. This typeof biopsy can be performed either from the sternum (breastbone) or theiliac crest hipbone (the bone area on either side of the pelvis on thelower back area). The skin is cleansed and a local anesthetic is givento numb the area. A long, rigid needle is inserted into the marrow, andcells are aspirated for study; this step is occasionally uncomfortable.A core biopsy (removing a small bone ‘chip’ from the marrow) may followthe aspiration.

In a further embodiment, an excisional or incisional biopsy can beperformed on the mammal. This type of biopsy is often used when a wideror deeper portion of the skin is needed. Using a scalpel (surgicalknife), a full thickness of skin is removed for further examination, andthe wound is sutured (sewed shut with surgical thread). When the entiretumor is removed, it is referred to as an excisional biopsy technique.If only a portion of the tumor is removed, it is referred to as anincisional biopsy technique. Excisional biopsy is often the methodusually preferred, for example, when melanoma (a type of skin cancer) issuspected.

In still further embodiments, a fine needle aspiration (FNA) biopsy canbe used. This type of biopsy involves using a thin needle to remove verysmall pieces from a tumor. Local anesthetic is sometimes used to numbthe area, but the test rarely causes much discomfort and leaves no scar.FNA is not, for example, used for diagnosis of a suspicious mole, butmay be used, for example, to biopsy large lymph nodes near a melanoma tosee if the melanoma has metastasized (spread). A computed tomographyscan (CT or CAT scan) can be used to guide a needle into a tumor in aninternal organ such as the lung or liver.

In other embodiments, punch shave and/or skin biopsies can be conducted.Punch biopsies involve taking a deeper sample of skin with a biopsyinstrument that removes a short cylinder, or “apple core,” of tissue.After a local anesthetic is administered, the instrument is rotated onthe surface of the skin until it cuts through all the layers, includingthe dermis, epidermis, and the most superficial parts of the subcutis(fat). A shave biopsy involves removing the top layers of skin byshaving it off. Shave biopsies are also performed with a localanesthetic. Skin biopsies involve removing a sample of skin forexamination under the microscope to determine if, for example, melanomais present. The biopsy is performed under local anesthesia.

In particular embodiment, methods are provided to determine whether thetumor overexpresses an Akt kinase. Akt kinase overexpression can referto the phosphorylation state of the kinase. Hyperphosphorylation of Aktcan be detected according to the methods described herein. In oneembodiment, a tumor biopsy can be compared to a control tissue. Thecontrol tissue can be a normal tissue from the mammal in which thebiopsy was obtained or a normal tissue from a healthy mammal Akt kinaseoverexpression or hyperphosphorylation can be determined if the tumorbiopsy contains greater amounts of Akt kinase and/or Akt kinasephosphorylation than the control tissue, such as, for example, at leastapproximately 1.5, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, 4, 4.25, 4.5,4.75, 5, 5.5, 6, 7, 8, 9, or 10-fold greater amounts of Akt kinase thancontained in the control tissue.

In one embodiment, the present invention provides a method to detectaberrant Akt kinase expression in a subject or in a biological samplefrom the subject by contacting cells, cell extracts, serum or othersample from the subjects or said biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formation, wherein an elevated presence ofthe complex relative to a normal cell is indicative of an aberrant cellthat expresses or overexpresses Akt. In one example, cells or cellextracts can be screened immunologically for the presence of elevatedlevels of Akt kinase.

In an alternative embodiment, the aberrant expression of Akt in a cellis detected at the genetic level by screening for the level ofexpression of a gene encoding an Akt kinase wherein an elevated level ofa transcriptional expression product (i.e., mRNA) compared to a normalcell is indicative of an aberrant cell. In certain embodiments,real-time PCR as well as other PCR procedures can be used to determinetranscriptional activity. In one embodiment, mRNA can be obtained fromcells of a subject or from a biological sample from a subject and cDNAoptionally generated. The mRNA or cDNA can then be contacted with agenetic probe capable of hybridizing to and/or amplifying all or part ofa nucleotide sequence encoding Akt kinase or its complementarynucleotide sequence and then the level of the mRNA or cDNA can bedetected wherein the presence of elevated levels of the mRNA or cDNAcompared to normal controls can be assessed.

Yet another embodiment of the present invention contemplates the use ofan antibody, monoclonal or polyclonal, to Akt kinase in a quantitativeor semi-quantitative diagnostic kit to determine relative levels of Aktkinase in suspected cancer cells from a patient, which can include allthe reagents necessary to perform the assay. In one embodiment, a kitutilizing reagents and materials necessary to perform an ELISA assay isprovided. Reagents can include, for example, washing buffer, antibodydilution buffer, blocking buffer, cell staining solution, developingsolution, stop solution, anti-phospho-protein specific antibodies,anti-Pan protein specific antibodies, secondary antibodies, anddistilled water. The kit can also include instructions for use and canoptionally be automated or semi-automated or in a form which iscompatible with automated machine or software. In one embodiment, aphosphor-ser-473 Akt antibody that detects the activated form of AKT(Akt phosphorylated at serine 474) can be utilized as the antibody in adiagnostic kit. See, for example, Yuan et al., Oncogene, 2000. 19: p.2324-2330.

5.6. AKT KINASES

Akt, also named PKB³, represents a subfamily of the serine/threoninekinase. Three members, AKT1, AKT2, and AKT3, have been identified inthis subfamily. Akt is activated by extracellular stimuli in aPI3K-dependent manner (Datta S. R. et al., Genes Dev, 1999. 13: p.2905-2927). Full activation of Akt requires phosphorylation of Thr³⁰⁸ inthe activation loop and Ser⁴⁷³ in the C-terminal activation domain. Aktis negatively regulated by PTEN tumor suppressor. Mutations in PTEN havebeen identified in various tumors, which lead to activation of Aktpathway (Datta S. R. et al., Genes Dev, 1999. 13: p. 2905-2927). Inaddition, amplification, overexpression and/or activation of Akt havebeen detected in a number of human malignancies (Datta S. R. et al.,Genes Dev, 1999. 13: p. 2905-2927); Cheng J. Q. and Nicosia S. V., inEncyclopedic Reference of Cancer, 2001, Schwab D. (ed.)Springer, BerlinHeidelberg and New York: pp 35-7). Ectopic expression of Akt, especiallyconstitutively active Akt, induces cell survival and malignanttransformation whereas inhibition of Akt activity stimulates apoptosisin a range of mammalian cells (Datta S. R. et al., Genes Dev, 1999. 13:p. 2905-2927); Cheng J. Q. and Nicosia S. V., in Encyclopedic Referenceof Cancer, 2001, Schwab D. (ed.)Springer, Berlin Heidelberg and NewYork: pp 35-7; Sun M. et al., Am J Path, 2001. 159: p. 431-437; Cheng J.Q. et al., Oncogene, 1997. 14: p. 2793-2801). Further, activation of Akthas been shown to associate with tumor invasiveness and chemoresistance(West K. A. et al., Drug Resist Updat, 2002. 5: p. 234-248).

Activation of the Akt pathway plays a pivotal role in malignanttransformation and chemoresistance by inducing cell survival, growth,migration, and angiogenesis. The present invention provides methods todetermine levels of Akt kinase overexpression and/or hyperactivated andphosphorylated Akt kinase.

The Akt kinase can be any known Akt family kinase, or kinase relatedthereto, including, but not limited to Akt 1, Akt 2, Akt 3. The mRNA andamino acid sequences of human Akt1, Akt2, and Akt 3 are illustrated inFIGS. 6 a-c, 7 a-d, and 8 a-c, respectively.

5.7. DIAGNOSTIC ASSAYS Immunological Assays

In one embodiment, a method is provided for detecting the aberrantexpression of an Akt kinase in a cell in a mammal or in a biologicalsample from the mammal, by contacting cells, cell extracts or serum orother sample from the mammal or biological sample with animmunointeractive molecule specific for an Akt kinase or antigenicportion thereof and screening for the level of immunointeractivemolecule-Akt kinase complex formations and determining whether anelevated presence of the complex relative to a normal cell is present.

The immunointeractive molecule can be a molecule having specificity andbinding affinity for an Akt kinase or its antigenic parts or itshomologs or derivatives thereof. In one embodiment, theimmunointeractive molecule can be an immunglobulin molecule. In otherembodiments, the immunointeractive molecules can be an antibodyfragments, single chain antibodies, and/or deimmunized moleculesincluding humanized antibodies and T-cell associated antigen-bindingmolecules (TABMs). In one particular embodiment, the antibody can be amonoclonal antibody. In another particular embodiment, the antibody canbe a polyclonal antibody. The immunointeractive molecule can exhibitspecificity for an Akt kinase or more particularly an antigenicdeterminant or epitope on an Akt kinase. An antigenic determinant orepitope on an Akt kinase includes that part of the molecule to which animmune response is directed. The antigenic determinant or epitope can bea B-cell epitope or where appropriate a T-cell epitope. In oneembodiment, the antibody is a phosphor-ser 473 Akt antibody.

One embodiment of the present invention provides a method for diagnosingthe presence of cancer or cancer-like growth in a mammal, in whichaberrant Akt activity is present, by contacting cells or cell extractsfrom the mammal or a biological sample from the subject with an Aktkinase-binding effective amount of an antibody having specificity forthe Akt kinase or an antigenic determinant or epitope thereon and thenquantitatively or qualitatively determining the level of an Aktkinase-antibody complex wherein the presence of elevated levels of saidcomplex compared to a normal cell is determined

Antibodies can be prepared by any of a number of means known to oneskilled in the art. For example, for the detection of human Akt kinase,antibodies can be generally but not necessarily derived from non-humananimals such as primates, livestock animals (e.g. sheep, cows, pigs,goats, horses), laboratory test animals (e.g. mice, rats, guinea pigs,rabbits) and/or companion animals (e.g. dogs, cats). Antibodies may alsobe recombinantly produced in prokaryotic or eukaryotic host cells.Generally, antibody based assays can be conducted in vitro on cell ortissue biopsies. However, if an antibody is suitably deimmunized or, inthe case of human use, humanized, then the antibody can be labeled with,for example, a nuclear tag, administered to a patient and the site ofnuclear label accumulation determined by radiological techniques. TheAkt kinase antibody can be a cancer targeting agent. Accordingly,another embodiment of the present invention provides deimmunized formsof the antibodies for use in cancer imaging in human and non-humanpatients.

In general, for the generation of antibodies to an Akt kinase, theenzyme is required to be extracted from a biological sample whether thisbe from animal including human tissue or from cell culture if producedby recombinant means. The Akt kinase can be separated from thebiological sample by any suitable means. For example, the separation maytake advantage of any one or more of the Akt kinase's surface chargeproperties, size, density, biological activity and its affinity foranother entity (e.g. another protein or chemical compound to which itbinds or otherwise associates). Thus, for example, separation of the Aktkinase from the biological fluid can be achieved by any one or more ofultra-centrifugation, ion-exchange chromatography (e.g. anion exchangechromatography, cation exchange chromatography), electrophoresis (e.g.polyacrylamide gel electrophoresis, isoelectric focussing), sizeseparation (e.g., gel filtration, ultra-filtration) andaffinity-mediated separation (e.g. immunoaffinity separation including,but not limited to, magnetic bead separation such as Dynabead(trademark) separation, immunochromatography, immuno-precipitation). Theseparation of Akt kinase from the biological fluid can preserveconformational epitopes present on the kinase and, thus, suitably avoidstechniques that cause denaturation of the enzyme. In a furtherembodiment, the kinase can be separated from the biological fluid usingany one or more of affinity separation, gel filtration and/orultra-filtration.

Immunization and subsequent production of monoclonal antibodies can becarried out using standard protocols known in the art, such as, forexample, described by Kohler and Milstein (Kohler and Milstein, Nature,1975. 256: p. 495-499; Kohler and Milstein, Eur J Immunol, 1976. 6(7):p. 511-519; Coligan et al., (Current Protocols in Immunology, 1991-1997,John Wiley & Sons, Inc.) or Toyama et al. (Monoclonal Antibody,Experiment Manual, 1987, Kodansha Scientific). Essentially, an animal isimmunized with an Akt kinase-containing biological fluid or fractionthereof or a recombinant form of Akt kinase by standard methods toproduce antibody-producing cells, particularly antibody-producingsomatic cells (e.g. B lymphocytes). These cells can then be removed fromthe immunized animal for immortalization. In certain embodiment, afragment of an Akt kinase can be used to the generate antibodies. Thefragment can be associated with a carrier. The carrier can be anysubstance of typically high molecular weight to which a non- or poorlyimmunogenic substance (e.g. a hapten) is naturally or artificiallylinked to enhance its immunogenicity.

Immortalization of antibody-producing cells can be carried out usingmethods which are well-known in the art. For example, theimmortalization may be achieved by the transformation method usingEpstein-Barr virus (EBV) (Kozbor et al., Methods in Enzymology, 1986.121: p. 140). In another embodiment, antibody-producing cells areimmortalized using the cell fusion method (described in Coligan et al.,1991-1997, supra), which is widely employed for the production ofmonoclonal antibodies. In this method, somatic antibody-producing cellswith the potential to produce antibodies, particularly B cells, arefused with a myeloma cell line. These somatic cells may be derived fromthe lymph nodes, spleens and peripheral blood of primed animals,preferably rodent animals such as mice and rats. In a particularembodiment, mice spleen cells can be used. In other embodiments, rat,rabbit, sheep or goat cells can also be used. Specialized myeloma celllines have been developed from lymphocytic tumours for use inhybridoma-producing fusion procedures (Kohler and Milstein, 1976, supra;Shulman et al., Nature, 1978. 276: p. 269-270; Volk et al., J Virol,1982. 42(1): p. 220-227). Many myeloma cell lines can also be used forthe production of fused cell hybrids, including, e.g. P3.times.63-Ag8,P3.times.63-AG8.653, P3/NS1-Ag4-1 (NS-1), Sp2/0-Ag14 and5194/5.XXO.Bu.1. The P3.times.63-Ag8 and NS-1 cell lines have beendescribed by Kohler and Milstein (1976, supra). Shulman et al. (1978,supra) developed the Sp2/0-Ag14 myeloma line. The S194/5.XXO.Bu.1 linewas reported by Trowbridge (J Exp Med, 1978. 148(1): p. 313-323).Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually involve mixing somatic cells withmyeloma cells in a 10:1 proportion (although the proportion may varyfrom about 20:1 to about 1:1), respectively, in the presence of an agentor agents (chemical, viral or electrical) that promotes the fusion ofcell membranes. Fusion methods have been described (Kohler and Milstein,1975, supra; Kohler and Milstein, 1976, supra; Gefter et al., SomaticCell Genet, 1977. 3: p. 231-236; Volk et al., 1982, supra). Thefusion-promoting agents used by those investigators were Sendai virusand polyethylene glycol (PEG). In certain embodiments, means to selectthe fused cell hybrids from the remaining unfused cells, particularlythe unfused myeloma cells, are provided. Generally, the selection offused cell hybrids can be accomplished by culturing the cells in mediathat support the growth of hybridomas but prevent the growth of theunfused myeloma cells, which normally would go on dividing indefinitely.The somatic cells used in the fusion do not maintain long-term viabilityin in vitro culture and hence do not pose a problem. Several weeks arerequired to selectively culture the fused cell hybrids. Early in thistime period, it is necessary to identify those hybrids which produce thedesired antibody, so that they may subsequently be cloned andpropagated. Generally, around 10% of the hybrids obtained produce thedesired antibody, although a range of from about 1 to about 30% is notuncommon. The detection of antibody-producing hybrids can be achieved byany one of several standard assay methods, including enzyme-linkedimmunoassay and radioimmunoassay techniques as, for example, describedin Kennet et al. (Monoclonal Antibodies and Hybridomas: A New Dimensionin Biological Analyses, 1980, Plenum Press, New York, p. 376-384) and byFACS analysis (O'Reilly et al., Biotechniques, 1998 25: p. 824-830).

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A suspension of the hybridomacells can be injected into a histocompatible animal. The injected animalwill then develop tumours that secrete the specific monoclonal antibodyproduced by the fused cell hybrid. The body fluids of the animal, suchas serum or ascites fluid, can be tapped to provide monoclonalantibodies in high concentration. Alternatively, the individual celllines may be propagated in vitro in laboratory culture vessels. Theculture medium containing high concentrations of a single specificmonoclonal antibody can be harvested by decantation, filtration orcentrifugation, and subsequently purified.

The cell lines can then be tested for their specificity to detect theAkt kinase of interest by any suitable immunodetection means. Forexample, cell lines can be aliquoted into a number of wells andincubated and the supernatant from each well is analyzed byenzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibodytechnique, or the like. The cell line(s) producing a monoclonal antibodycapable of recognizing the target LIM kinase but which does notrecognize non-target epitopes are identified and then directly culturedin vitro or injected into a histocompatible animal to form tumours andto produce, collect and purify the required antibodies.

The present invention provides, therefore, a method of detecting in asample an Akt kinase or fragment, variant or derivative thereofcomprising contacting the sample with an antibody or fragment orderivative thereof and detecting the level of a complex containing theantibody and Akt kinase or fragment, variant or derivative thereofcompared to normal controls wherein elevated levels of Akt kinase isdetermined Any suitable technique for determining formation of thecomplex may be used. For example, an antibody according to theinvention, having a reporter molecule associated therewith, may beutilized in immunoassays. Such immunoassays include but are not limitedto radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs)immunochromatographic techniques (ICTs), and Western blotting which arewell known to those of skill in the art. Immunoassays can also includecompetitive assays. The present invention encompasses qualitative andquantitative immunoassays.

Suitable immunoassay techniques are described, for example, in U.S. Pat.Nos. 4,016,043; 4,424,279; and 4,018,653. These include both single-siteand two-site assays of the non-competitive types, as well as thetraditional competitive binding assays. These assays also include directbinding of a labeled antigen-binding molecule to a target antigen.

The invention further provides methods for quantifying Akt proteinexpression and activation levels in cells or tissue samples obtainedfrom an animal, such as a human cancer patient or an individualsuspected of having cancer. In one embodiment, the invention providesmethods for quantifying Akt protein expression or activation levelsusing an imaging system quantitatively. The imaging system can be usedto receive, enhance, and process images of cells or tissue samples, thathave been stained with AKT protein-specific stains, in order todetermine the amount or activation level of AKT protein expressed in thecells or tissue samples from such an animal. In embodiments of themethods of the invention, a calibration curve of AKT1 and AKT2 proteinexpression can be generated for at least two cell lines expressingdiffering amounts of AKT protein. The calibration curve can then used toquantitatively determine the amount of AKT protein that is expressed ina cell or tissue sample. Analogous calibration curves can be made foractivated AKT proteins using reagents specific for the activationfeatures. It can also be used to determine changes in amounts andactivation state of AKT before and after clinical cancer treatment.

In one particular embodiment of the methods of the invention, AKTprotein expression in a cell or tissue sample can be quantified using anenzyme-linked immunoabsorbent assay (ELISA) to determine the amount ofAKT protein in a sample. Such methods are described, for example, inU.S. Patent Publication No. 2002/0015974.

In other embodiments enzyme immunoassays can be used to detect the Aktkinase. In such assays, an enzyme is conjugated to the second antibody,generally by means of glutaraldehyde or periodate. The substrates to beused with the specific enzymes are generally chosen for the productionof, upon hydrolysis by the corresponding enzyme, a detectable colourchange. It is also possible to employ fluorogenic substrates, whichyield a fluorescent product rather than the chromogenic substrates. Theenzyme-labeled antibody can be added to the first antibody-antigencomplex, allowed to bind, and then the excess reagent washed away. Asolution containing the appropriate substrate can then be added to thecomplex of antibody-antigen-antibody. The substrate can react with theenzyme linked to the second antibody, giving a qualitative visualsignal, which may be further quantitated, usuallyspectrophotometrically, to give an indication of the amount of antigenwhich was present in the sample. Alternately, fluorescent compounds,such as fluorescein, rhodamine and the lanthanide, europium (EU), can bechemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic colour visually detectable with a lightmicroscope. The fluorescent-labeled antibody is allowed to bind to thefirst antibody-antigen complex. After washing off the unbound reagent,the remaining tertiary complex is then exposed to light of anappropriate wavelength. The fluorescence observed indicates the presenceof the antigen of interest. Immunofluorometric assays (IFMA) are wellestablished in the art and are particularly useful for the presentmethod. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules can also be employed.

In a particular embodiment, antibodies to Akt kinase can also be used inELISA-mediated detection of Akt kinase especially in serum or othercirculatory fluid. This can be accomplished by immobilizing anti-Aktkinase antibodies to a solid support and contacting these with abiological extract such as serum, blood, lymph or other bodily fluid,cell extract or cell biopsy. Labeled anti-Akt kinase antibodies can thenbe used to detect immobilized Akt kinase. This assay can be varied inany number of ways and all variations are encompassed by the presentinvention and known to one skilled in the art. This approach can enablerapid detection and quantitation of Akt kinase levels using, forexample, a serum-based assay.

In one embodiment, an Akt ELISA assay kit may be used in the presentinvention. For example, a Cellular Activation of Signaling ELISA kit forAkt S473 from SuperArray Bioscience can be utilized in the presentinvention. In one embodiment, the antibody can be an anti-pan antibodythat recognizes Akt S473. ELISA assay kit containing an anti-Aktantibody and additional reagents, including, but not limited to, washingbuffer, antibody dilution buffer, blocking buffer, cell stainingsolution, developing solution, stop solution, secondary antibodies, anddistilled water.

Nucleotide Detection

In another embodiment, a method to detect Akt kinases is provided bydetecting the level of expression in a cell of a polynucleotide encodingan Akt kinase. Expression of the polynucleotide can be determined usingany suitable technique known to one skilled in the art. In oneembodiment, a labeled polynucleotide encoding an Akt kinase can beutilized as a probe in a Northern blot of an RNA extract obtained fromthe cell. In other embodiments, a nucleic acid extract from an animalcan be utilized in concert with oligonucleotide primers corresponding tosense and antisense sequences of a polynucleotide encoding the kinase,or flanking sequences thereof, in a nucleic acid amplification reactionsuch as RT PCR. A variety of automated solid-phase detection techniquesare also available to one skilled in the art, for example, as describedby Fodor et al. (Science, 1991. 251: p. 767-777) and Kazal et al.(Nature Medicine, 1996. 2: p. 753-759).

In other embodiments, methods are provided to detect Akt kinase encodingRNA transcripts. The RNA can be isolated from a cellular samplesuspected of containing Akt kinase RNA, e.g. total RNA isolated fromhuman cancer tissue. RNA can be isolated by methods known in the art,e.g. using TRIZOL reagent (GIBCO-BRL/Life Technologies, Gaithersburg,Md.). Oligo-dT, or random-sequence oligonucleotides, as well assequence-specific oligonucleotides can be employed as a primer in areverse transcriptase reaction to prepare first-strand cDNAs from theisolated RNA. Resultant first-strand cDNAs can then amplified withsequence-specific oligonucleotides in PCR reactions to yield anamplified product.

Polymerase chain reaction or “PCR” refers to a procedure or technique inwhich amounts of a preselected fragment of nucleic acid, RNA and/or DNA,are amplified as described, for example, in U.S. Pat. No. 4,683,195.Generally, sequence information from the ends of the region of interestor beyond is employed to design oligonucleotide primers. These primerswill be identical or similar in sequence to opposite strands of thetemplate to be amplified. PCR can be used to amplify specific RNAsequences and cDNA transcribed from total cellular RNA. See generallyMullis et al. (Quant Biol, 1987. 51: p. 263; Erlich, eds., PCRTechnology, 1989, Stockton Press, NY). Thus, amplification of specificnucleic acid sequences by PCR relies upon oligonucleotides or “primers”having conserved nucleotide sequences wherein the conserved sequencesare deduced from alignments of related gene or protein sequences, e.g. asequence comparison of mammalian Akt kinase genes. For example, oneprimer is prepared which is predicted to anneal to the antisense strandand another primer prepared which is predicted to anneal to the sensestrand of a cDNA molecule which encodes a Akt kinase. To detect theamplified product, the reaction mixture is typically subjected toagarose gel electrophoresis or other convenient separation technique andthe relative presence of the Akt kinase specific amplified DNA detected.For example, Akt kinase amplified DNA may be detected using Southernhybridization with a specific oligonucleotide probe or comparing itselectrophoretic mobility with DNA standards of known molecular weight.Isolation, purification and characterization of the amplified Akt kinaseDNA can be accomplished by excising or eluting the fragment from the gel(for example, see references Lawn et al., Nucleic Acids Res, 1981. 2: p.6103; Goeddel et al., Nucleic Acids Res, 1980. 8: p. 4057), cloning theamplified product into a cloning site of a suitable vector, such as thepCRII vector (Invitrogen), sequencing the cloned insert and comparingthe DNA sequence to the known sequence of LIM kinase. The relativeamounts of LIM kinase mRNA and cDNA can then be determined

In one embodiment, real-time PCR can be used to determinetranscriptional levels of Akt nucleotides. Determination oftranscriptional activity also includes a measure of potentialtranslational activity based on available mRNA transcripts. Real-timePCR as well as other PCR procedures use a number of chemistries fordetection of PCR product including the binding of DNA bindingfluorophores, the 5′ endonuclease, adjacent liner and hairpinoligoprobes and the self-fluorescing amplicons. These chemistries andreal-time PCR in general are discussed, for example, in Mackay et al.,Nucleic Acids Res, 2002. 30(6): p. 1292-1305; Walker, J Biochem MolToxicology, 2001. 15(3): p. 121-127; Lewis et al., J Pathol, 2001. 195:p. 66-71.

In an alternate embodiment, the aberrant expression of Akt can beidentified by contacting a nucleotide sequences isolated from abiological sample with an oligonucleotide probe having a sequencecomplementary to an Akt sequences selected from the nucleotide sequencesof FIGS. 6 a-c, 7 a-d, or 8 a-c, or fragment thereof, and then detectingthe sequence by hybridizing the probe to the sequence, and comparing theresults to a normal sample. The hybridization of the probe to thebiological sample can be detected by labeling the probe using anydetectable agent. The probe can be labeled for example, with aradioisotope, or with biotin, fluorescent dye, electron-dense reagent,enzyme, hapten or protein for which antibodies are available. Thedetectable label can be assayed by any desired means, includingspectroscopic, photochemical, biochemical, immunochemical,radioisotopic, or chemical means. The probe can also be detected usingtechniques such as an oligomer restriction technique, a dot blot assay,a reverse dot blot assay, a line probe assay, and a 5′ nuclease assay.Alternatively, the probe can be detected using any of the generallyapplicable DNA array technologies, including macroarray, microarray andDNA microchip technologies. The oligonucleotide probe typically includesapproximately at least 14, 15, 16, 18, 20, or 28 nucleotides thathybridize to the nucleotides selected from FIGS. 6 a-c, 7 a-d, and 8a-c, or a fragment thereof. It is generally not preferred to use a probethat is greater than approximately 25 or 28 nucleotides in length. Theoligonucleotide probe is designed to identify an Akt nucleotidesequence.

Kinase Assays

The activity of the Akt kinases can be measured using any suitablekinase assay known in the art. For example, and not by way oflimitation, the methods described in Hogg et al (Oncogene, 1994. 9: p.98-96), Mills et al (J Biol Chem, 1992. 267: p. 16000-006) and Tomizawaet al. (FEBS Lett, 2001. 492: p. 221-7), Schmandt et al. (J Immunol,1994. 152: p. 96-105) can be used. Further serine, threonine andtyrosine kinase assays are described in Ausubel et al. (Short Protocolsin Molecular Biology, 1999, unit 17.6).

Akt kinase assays can generally use an Akt polypeptide, a labeled donorsubstrate, and a receptor substrate that is either specific ornon-specific for Akt. In such assays Akt transfers a labeled moiety fromthe donor substrate to the receptor substrate, and kinase activity ismeasured by the amount of labeled moiety transferred from the donorsubstrate to the receptor substrate. Akt polypeptide can be producedusing various expression systems, can be purified from cells, can be inthe form of a cleaved or uncleaved recombinant fusion protein and/or canhave non-Akt polypeptide sequences, for example a His tag or.beta.-galactosidase at its N- or C-terminus Akt activity can be assayedin cancerous cells lines if the cancerous cell lines are used as asource of the Akt to be assayed. Suitable donor substrates for Aktassays include any molecule that is susceptible to dephosphorylation byAkt., such as, for example include .gamma-labeled ATP and ATP analogs,wherein the label is ³³P, ³²P, ³⁵S or any other radioactive isotope or asuitable fluorescent marker. Suitable recipient substrates for Aktassays include any polypeptide or other molecule that is susceptible tophosphorylation by Akt. Recipient substrates can be derived fromfragments of in vivo targets of Akt. Recipient substrates fragments canbe 8 to 50 amino acids in length, usually 10 to 30 amino acids andparticularly of about 10, 12, 15, 18, 20 and 25 amino acids in length.Further recipient substrates can be determined empirically using a setof different polypeptides or other molecules. Targets of Recipientsubstrates for TTK can be capable of being purified from othercomponents of the reaction once the reaction has been performed. Thispurification is usually done through a molecular interaction, where therecipient substrates is biotinylated and purified through itsinteraction with streptavidin, or a specific antibody is available thatcan specifically recognize the recipient substrates. The reaction can beperformed in a variety of conditions, such as on a solid support, in agel, in solution or in living cells. The choice of detection methodsdepends on type of label used for the donor molecule and may include,for example, measurement of incorporated radiation or fluorescence byautoradiography, scintillation, scanning or fluorography.

6. METHODS OF TREATMENT

The compounds and pharmaceutical compositions provided herein can beused in the treatment of a condition including tumors, cancer, and otherdisorders associated with abnormal cell proliferation. In oneembodiment, the compounds of the present invention can be used to treata carcinoma, sarcoma, lymphoma, leukemia, and/or myeloma. In otherembodiments of the present invention, the compounds disclosed herein canbe used to treat solid tumors.

The compounds of the present invention invention can be used for thetreatment of cancer, such as, but not limited to cancer of the followingorgans or tissues: breast, prostate, lung, bronchus, colon, urinary,bladder, non-Hodgkin lymphoma, melanoma, kidney, renal, pancreas,pharnx, thyroid, stomach, brain, multiple myeloma, esophagus, liver,intrahepatic bile duct, cervix, larynx, acute myeloid leukemia, chroniclymphatic leukemia, soft tissue, such as heart, Hodgkin lymphoma,testis, small intestine, chronic myeloid leukemia, acute lymphaticleukemia, anus, anal canal, anorectal, thyroid, vulva, gallbladder,pleura, eye, nose nasal cavity, middle ear, nasopharnx, ureter,peritoneum, omentum, mesentery, and gastrointestineal, high gradeglioma, glioblastoma, colon, rectal, pancreatic, gastric cancers,hepatocellular carcinoma; head and neck cancers, carcinomas; renal cellcarcinoma; adenocarcinoma; sarcomas; hemangioendothelioma; lymphomas;leukemias, mycosis fungoides. In additional embodiments, the compoundsof the present invention can be used to treat skin diseases including,but not limited to, the malignant diseases angiosarcoma,hemangioendothelioma, basal cell carcinoma, squamous cell carcinoma,malignant melanoma and Kaposi's sarcoma, and the non-malignant diseasesor conditions such as psoriasis, lymphangiogenesis, hemangioma ofchildhood, Sturge-Weber syndrome, verruca vulgaris, neurofibromatosis,tuberous sclerosis, pyogenic granulomas, recessive dystrophicepidermolysis bullosa, venous ulcers, acne, rosacea, eczema, molluscumcontagious, seborrheic keratosis, and actinic keratosis.

Compositions including the compounds of the invention can be used totreat these cancers and other cancers at any stage from the discovery ofthe cancer to advanced stages. In addition, compositions includingcompounds of the invention can be used in the treatment of the primarycancer and metastases thereof.

In other embodiments of the invention, the compounds described hereincan be used for the treatment of cancer, including, but not limited tothe cancers listed in Table 2 below.

TABLE 2 Types of Cancer Acute Lymphoblastic Leukemia, Adult AcuteLymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Adult AcuteMyeloid Leukemia, Childhood Adrenocortical Carcinoma AdrenocorticalCarcinoma, Childhood AIDS-Related Cancers AIDS-Related Lymphoma AnalCancer Astrocytoma, Childhood Cerebellar Astrocytoma, Childhood CerebralBasal Cell Carcinoma Bile Duct Cancer, Extrahepatic Bladder CancerBladder Cancer, Childhood Bone Cancer, Osteosarcoma/Malignant FibrousHistiocytoma Brain Stem Glioma, Childhood Brain Tumor, Adult BrainTumor, Brain Stem Glioma, Childhood Brain Tumor, Cerebellar Astrocytoma,Childhood Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, ChildhoodBrain Tumor, Ependymoma, Childhood Brain Tumor, Medulloblastoma,Childhood Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors,Childhood Brain Tumor, Visual Pathway and Hypothalamic Glioma, ChildhoodBrain Tumor, Childhood Breast Cancer Breast Cancer, Childhood BreastCancer, Male Bronchial Adenomas/Carcinoids, Childhood Burkitt's LymphomaCarcinoid Tumor, Childhood Carcinoid Tumor, Gastrointestinal Carcinomaof Unknown Primary Central Nervous System Lymphoma, Primary CerebellarAstrocytoma, Childhood Cerebral Astrocytoma/Malignant Glioma, ChildhoodCervical Cancer Childhood Cancers Chronic Lymphocytic Leukemia ChronicMyelogenous Leukemia Chronic Myeloproliferative Disorders Colon CancerColorectal Cancer, Childhood Cutaneous T-Cell Lymphoma, see MycosisFungoides and Sézary Syndrome Endometrial Cancer Ependymoma, ChildhoodEsophageal Cancer Esophageal Cancer, Childhood Ewing's Family of TumorsExtracranial Germ Cell Tumor, Childhood Extragonadal Germ Cell TumorExtrahepatic Bile Duct Cancer Eye Cancer, Intraocular Melanoma EyeCancer, Retinoblastoma Gallbladder Cancer Gastric (Stomach) CancerGastric (Stomach) Cancer, Childhood Gastrointestinal Carcinoid TumorGerm Cell Tumor, Extracranial, Childhood Germ Cell Tumor, ExtragonadalGerm Cell Tumor, Ovarian Gestational Trophoblastic Tumor Glioma, AdultGlioma, Childhood Brain Stem Glioma, Childhood Cerebral AstrocytomaGlioma, Childhood Visual Pathway and Hypothalamic Skin Cancer (Melanoma)Skin Carcinoma, Merkel Cell Small Cell Lung Cancer Small IntestineCancer Soft Tissue Sarcoma, Adult Soft Tissue Sarcoma, ChildhoodSquamous Cell Carcinoma, see Skin Cancer (non-Melanoma) Squamous NeckCancer with Occult Primary, Metastatic Stomach (Gastric) Cancer Stomach(Gastric) Cancer, Childhood Supratentorial Primitive NeuroectodermalTumors, Childhood T-Cell Lymphoma, Cutaneous, see Mycosis Fungoides andSézary Syndrome Testicular Cancer Thymoma, Childhood Thymoma and ThymicCarcinoma Thyroid Cancer Thyroid Cancer, Childhood Transitional CellCancer of the Renal Pelvis and Ureter Trophoblastic Tumor, GestationalUnknown Primary Site, Carcinoma of, Adult Unknown Primary Site, Cancerof, Childhood Unusual Cancers of Childhood Ureter and Renal Pelvis,Transitional Cell Cancer Urethral Cancer Uterine Cancer, EndometrialUterine Sarcoma Vaginal Cancer Visual Pathway and Hypothalamic Glioma,Childhood Vulvar Cancer Waldenström's Macroglobulinemia Wilms' TumorHairy Cell Leukemia Head and Neck Cancer Hepatocellular (Liver) Cancer,Adult (Primary) Hepatocellular (Liver) Cancer, Childhood (Primary)Hodgkin's Lymphoma, Adult Hodgkin's Lymphoma, Childhood Hodgkin'sLymphoma During Pregnancy Hypopharyngeal Cancer Hypothalamic and VisualPathway Glioma, Childhood Intraocular Melanoma Islet Cell Carcinoma(Endocrine Pancreas) Kaposi's Sarcoma Kidney (Renal Cell) Cancer KidneyCancer, Childhood Laryngeal Cancer Laryngeal Cancer, Childhood Leukemia,Acute Lymphoblastic, Adult Leukemia, Acute Lymphoblastic, ChildhoodLeukemia, Acute Myeloid, Adult Leukemia, Acute Myeloid, ChildhoodLeukemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, BCell Lip and Oral Cavity Cancer Liver Cancer, Adult (Primary) LiverCancer, Childhood (Primary) Lung Cancer, Non-Small Cell Lung Cancer,Small Cell Lymphoma, AIDS-Related Lymphoma, Burkitt's Lymphoma,Cutaneous T-Cell, see Mycosis Fungoides and Sézary Syndrome Lymphoma,Hodgkin's, Adult Lymphoma, Hodgkin's, Childhood Lymphoma, Hodgkin'sDuring Pregnancy Lymphoma, Non-Hodgkin's, Adult Lymphoma, Non-Hodgkin's,Childhood Lymphoma, Non-Hodgkin's During Pregnancy Lymphoma, PrimaryCentral Nervous System Macroglobulinemia, Waldenström's MalignantFibrous Histiocytoma of Bone/Osteosarcoma Medulloblastoma, ChildhoodMelanoma Melanoma, Intraocular (Eye) Merkel Cell Carcinoma Mesothelioma,Adult Malignant Mesothelioma, Childhood Metastatic Squamous Neck Cancerwith Occult Primary Multiple Endocrine Neoplasia Syndrome, ChildhoodMultiple Myeloma/Plasma Cell Neoplasm Mycosis Fungoides MyelodysplasticSyndromes Myelodysplastic/Myeloproliferative Diseases MyelogenousLeukemia, Chronic Myeloid Leukemia, Adult Acute Myeloid Leukemia,Childhood Acute Myeloma, Multiple Myeloproliferative Disorders, ChronicNasal Cavity and Paranasal Sinus Cancer Nasopharyngeal CancerNasopharyngeal Cancer, Childhood Neuroblastoma Non-Hodgkin's Lymphoma,Adult Non-Hodgkin's Lymphoma, Childhood Non-Hodgkin's Lymphoma DuringPregnancy Non-Small Cell Lung Cancer Oral Cancer, Childhood Oral CavityCancer, Lip and Oropharyngeal Cancer Osteosarcoma/Malignant FibrousHistiocytoma of Bone Ovarian Cancer, Childhood Ovarian Epithelial CancerOvarian Germ Cell Tumor Ovarian Low Malignant Potential Tumor PancreaticCancer Pancreatic Cancer, Childhood Pancreatic Cancer, Islet CellParanasal Sinus and Nasal Cavity Cancer Parathyroid Cancer Penile CancerPheochromocytoma Pineoblastoma and Supratentorial PrimitiveNeuroectodermal Tumors, Childhood Pituitary Tumor Plasma CellNeoplasm/Multiple Myeloma Pleuropulmonary Blastoma Pregnancy and BreastCancer Pregnancy and Hodgkin's Lymphoma Pregnancy and Non-Hodgkin'sLymphoma Primary Central Nervous System Lymphoma Prostate Cancer RectalCancer Renal Cell (Kidney) Cancer Renal Cell (Kidney) Cancer, ChildhoodRenal Pelvis and Ureter, Transitional Cell Cancer RetinoblastomaRhabdomyosarcoma, Childhood Salivary Gland Cancer Salivary Gland Cancer,Childhood Sarcoma, Ewing's Family of Tumors Sarcoma, Kaposi's Sarcoma,Soft Tissue, Adult Sarcoma, Soft Tissue, Childhood Sarcoma, UterineSezary Syndrome Skin Cancer (non-Melanoma) Skin Cancer, Childhood

In further embodiments of the present invention, the compounds disclosedherein can be used in the treatment of angiogenesis-related diseases.

Antiangiogenic small molecules include thalidomide, which acts in partby inhibiting NFkB, 2-methoxyestradiol, which influences microtubuleactivation and hypoxia inducing factor (HIF1a) activation,cyclo-oxygenase 2 (COX2) inhibitors, and low doses of conventionalchemotherapeutic agents, including cyclophosphamide, taxanes, and vincaalkaloids (vincristine, vinblastine) (D'Amato R. J. et al., Proc. Natl.Acad. Sci. U.S.A., 1994. 91: p. 3964-3968; D'Amato R. J. et al., Proc.Natl. Acad. Sci. U.S.A., 1994. 91: p. 4082-4085). In addition, certaintyrosine kinase inhibitors indirectly decrease angiogenesis bydecreasing production of VEGF and other proangiogenic factors by tumorand stromal cells. These drugs include Herceptin, imatinib (Glivec), andIressa (Bergers G. et al., J Clin Invest, 2003. 111: p. 1287-1295;Ciardiello F. et al., Clin Cancer Res, 2001. 7: p. 1459-1465; Plum S. M.et al., Clin Cancer Res, 2003. 9: p. 4619-4626).

Recently, angiogenesis inhibitors have moved from animal models to humanpatients. Angiogenesis inhibitors represent a promising treatment for avariety of cancers. Recently, Avastin a high affinity antibody againstvascular endothelial growth factor (VEGF), has been shown to prolonglife as a single agent in advanced renal cell carcinoma and prolong lifein combination with chemotherapy in advanced colon cancer (Yang J. C. etal., New Eng J Med, 2003. 349: p. 427-434; Kabbinavar F. et al., J ClinOne, 2003. 21: p. 60-65).

Angiogenesis-related diseases include, but are not limited to,inflammatory, autoimmune, and infectous diseases; angiogenesis-dependentcancer, including, for example, solid tumors, blood born tumors such asleukemias, and tumor metastases; benign tumors, for example hemangiomas,acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas;rheumatoid arthritis; psoriasis; eczema; ocular angiogenic diseases, forexample, diabetic retinopathy, retinopathy of prematurity, maculardegeneration, corneal graft rejection, neovascular glaucoma, retrolentalfibroplasia, rubeosis; Osler-Webber Syndrome; myocardial angiogenesis;plaque neovascularization; telangiectasia; hemophiliac joints;angiofibroma; and wound granulation. In addition, compositions of thisinvention can be used to treat diseases such as, but not limited to,intestinal adhesions, atherosclerosis, scleroderma, warts, andhypertrophic scars (i.e., keloids). Compositions of this invention canalso be used in the treatment of diseases that have angiogenesis as apathologic consequence such as cat scratch disease (Rochele minaliaquintosa), ulcers (Helobacter pylori), tuberculosis, and leprosy.

6.1. TREATMENT OF DRUG RESISTANT TUMORS OR CANCERS

The invention provides compounds that can be used to treat drugresistant cancer, including the embodiments of cancers and thetriciribine compound and/or the a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof.

Multidrug resistance (MDR) occurs in human cancers and can be asignificant obstacle to the success of chemotherapy. Multidrugresistance is a phenomenon whereby tumor cells in vitro that have beenexposed to one cytotoxic agent develop cross-resistance to a range ofstructurally and functionally unrelated compounds. In addition, MDR canoccur intrinsically in some cancers without previous exposure tochemotherapy agents. Thus, in one embodiment, the present inventionprovides methods for the treatment of a patient with a drug resistantcancer, for example, multidrug resistant cancer, by administration ofTCN, TCN-P or a related compound and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof.In certain embodiments, TCN, TCN-P and related compounds and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, can be used to treat cancers that are resistant totaxol alone, rapamycin, tamoxifen, cisplatin, and/or gefitinib (iressa).

In one embodiment, TCN, TCN-P or a related compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can be used for the treatment of drug resistant cancers ofthe colon, bone, kidney, adrenal, pancreas, liver and/or any othercancer known in the art or described herein.

6.2. COMBINATION THERAPY

In one embodiment, the triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can be administered together with other cytotoxic agents.In another embodiment, the triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, and compositions thereof, when used in the treatment ofsolid tumors, can be administered the use of radiation.

In another embodiment of the present invention, the triciribinecompounds and a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab or a salt thereof, and compositions disclosedherein can be combined with at least one additional chemotherapeuticagent. The additional agents can be administered in combination oralternation with the compounds disclosed herein. The drugs can form partof the same composition, or be provided as a separate composition foradministration at the same time or a different time.

In one embodiment, the triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, disclosed herein can be combined with antiangiogenicagents to enhance their effectiveness, or combined with otherantiangiogenic agents and administered together with other cytotoxicagents. In another embodiment, the triciribine compounds and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, and compositions, when used in the treatment of solidtumors, can be administered with the agents selected from, but notlimited to IL-12, retinoids, interferons, angiostatin, endostatin,thalidomide, thrombospondin-1, thrombospondin-2, captopryl,anti-neoplastic agents such as alpha interferon, COMP (cyclophosphamide,vincristine, methotrexate and prednisone), etoposide, mBACOD(methortrexate, bleomycin, doxorubicin, cyclophosphamide, vincristineand dexamethasone), PRO-MACE/MOPP (prednisone, methotrexate (w/leucovinrescue), doxorubicin, cyclophosphamide, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG andradiation. In further embodiments, the compounds and compositionsdisclosed herein can be administered in combination or alternation with,for example, drugs with antimitotic effects, such as those which targetcytoskeletal elements, including podophylotoxins or vinca alkaloids(vincristine, vinblastine); antimetabolite drugs (such as5-fluorouracil, cytarabine, gemcitabine, purine analogues such aspentostatin, methotrexate); alkylating agents or nitrogen mustards (suchas nitrosoureas, cyclophosphamide or ifosphamide); drugs which targetDNA such as the antracycline drugs adriamycin, doxorubicin,pharmorubicin or epirubicin; drugs which target topoisomerases such asetoposide; hormones and hormone agonists or antagonists such asestrogens, antiestrogens (tamoxifen and related compounds) andandrogens, flutamide, leuprorelin, goserelin, cyprotrone or octreotide;drugs which target signal transduction in tumour cells includingantibody derivatives such as herceptin; alkylating drugs such asplatinum drugs (cis-platin, carbonplatin, oxaliplatin, paraplatin) ornitrosoureas; drugs potentially affecting metastasis of tumours such asmatrix metalloproteinase inhibitors; gene therapy and antisense agents;antibody therapeutics; other bioactive compounds of marine origin,notably the didemnins such as aplidine; steroid analogues, in particulardexamethasone; anti-inflammatory drugs, including nonsteroidal agents(such as acetaminophen or ibuprofen) or steroids and their derivativesin particular dexamethasone; anti-emetic drugs, including 5HT-3inhibitors (such as gramisetron or ondasetron), and steroids and theirderivatives in particular dexamethasone. In still further embodiments,the compounds and compositions can be used in combination or alternationwith the chemotherapeutic agents disclosed below in Table 3.

TABLE 3 Chemotherapeutic Agents 13-cis-Retinoic Acid2-Amino-6-Mercaptopurine 2-CdA 2-Chlorodeoxyadenosine 5-fluorouracil5-FU 6-TG 6-Thioguanine 6-Mercaptopurine 6-MP Accutane Actinomycin-DAdriamycin Adrucil Agrylin Ala-Cort Aldesleukin Alemtuzumab AlitretinoinAlkaban-AQ Alkeran All-transretinoic acid Alpha interferon AltretamineAmethopterin Amifostine Aminoglutethimide Anagrelide AnandronAnastrozole Arabinosylcytosine Ara-C Aranesp Aredia Arimidex AromasinArsenic trioxide Asparaginase ATRA Avastin BCG BCNU BevacizumabBexarotene Bicalutamide BiCNU Blenoxane Bleomycin Bortezomib BusulfanBusulfex C225 Calcium Leucovorin Campath Camptosar Camptothecin-11Capecitabine Carac Carboplatin Carmustine Carmustine wafer Casodex CCNUCDDP CeeNU Cerabidine cetuximab Chlorambucil Cisplatin Citrovorum FactorCladribine Cortisone Cosmegen CPT-11 Cyclophosphamide CytadrenCytarabine Cytarabine liposomal Cytosar-U Cytoxan DacarbazineDactinomycin Darbepoetin alfa Daunomycin Daunorubicin Daunorubicinhydrochloride Daunorubicin liposomal DaunoXome Decadron Delta-CortefDeltasone Denileukin diftitox DepoCyt Dexamethasone Dexamethasoneacetate dexamethasone sodium phosphate Dexasone Dexrazoxane DHAD DICDiodex Docetaxel Doxil Doxorubicin Doxorubicin liposomal Droxia DTICDTIC-Dome Duralone Efudex Eligard Ellence Eloxatin Elspar EmcytEpirubicin Epoetin alfa Erbitux Erwinia L-asparaginase EstramustineEthyol Etopophos Etoposide Etoposide phosphate Eulexin Evista ExemestaneFareston Faslodex Femara Filgrastim Floxuridine Fludara FludarabineFluoroplex Fluorouracil Fluorouracil (cream) Fluoxymesterone FlutamideFolinic Acid FUDR Fulvestrant G-CSF Gefitinib Gemcitabine Gemtuzumabozogamicin Gemzar Gleevec Lupron Lupron Depot Matulane MaxidexMechlorethamine Mechlorethamine Hydrochlorine Medralone Medrol MegaceMegestrol Megestrol Acetate Melphalan Mercaptopurine Mesna MesnexMethotrexate Methotrexate Sodium Methylprednisolone Mylocel LetrozoleNeosar Neulasta Neumega Neupogen Nilandron Nilutamide Nitrogen MustardNovaldex Novantrone Octreotide Octreotide acetate Oncospar Oncovin OntakOnxal Oprevelkin Orapred Orasone Oxaliplatin Paclitaxel PamidronatePanretin Paraplatin Pediapred PEG Interferon Pegaspargase PegfilgrastimPEG-INTRON PEG-L-asparaginase Phenylalanine Mustard Platinol Platinol-AQPrednisolone Prednisone Prelone Procarbazine PROCRIT ProleukinProlifeprospan 20 with Carmustine implant Purinethol RaloxifeneRheumatrex Rituxan Rituximab Roveron-A (interferon alfa-2a) RubexRubidomycin hydrochloride Sandostatin Sandostatin LAR SargramostimSolu-Cortef Solu-Medrol STI-571 Streptozocin Tamoxifen Targretin TaxolTaxotere Temodar Temozolomide Teniposide TESPA Thalidomide ThalomidTheraCys Thioguanine Thioguanine Tabloid Thiophosphoamide ThioplexThiotepa TICE Toposar Topotecan Toremifene Trastuzumab Tretinoin TrexallTrisenox TSPA VCR Velban Velcade VePesid Vesanoid Viadur VinblastineVinblastine Sulfate Vincasar Pfs Vincristine Vinorelbine Vinorelbinetartrate VLB VP-16 Vumon Xeloda Zanosar Zevalin Zinecard ZoladexZoledronic acid Zometa Gliadel wafer Glivec GM-CSF Goserelingranulocyte - colony stimulating factor Granulocyte macrophage colonystimulating factor Halotestin Herceptin Hexadrol HexalenHexamethylmelamine HMM Hycamtin Hydrea Hydrocort Acetate HydrocortisoneHydrocortisone sodium phosphate Hydrocortisone sodium succinateHydrocortone phosphate Hydroxyurea Ibritumomab Ibritumomab TiuxetanIdamycin Idarubicin Ifex IFN-alpha Ifosfamide IL-2 IL-11 Imatinibmesylate Imidazole Carboxamide Interferon alfa Interferon Alfa-2b (PEGconjugate) Interleukin-2 Interleukin-11 Intron A (interferon alfa-2b)Leucovorin Leukeran Leukine Leuprolide Leurocristine Leustatin LiposomalAra-C Liquid Pred Lomustine L-PAM L-Sarcolysin Meticorten MitomycinMitomycin-C Mitoxantrone M-Prednisol MTC MTX Mustargen Mustine MutamycinMyleran Iressa Irinotecan Isotretinoin Kidrolase Lanacort L-asparaginaseLCR

In certain embodiments, interferons (IFNs) can be used in combinationswith the compounds of the present invention. Suitable intereferonsinclude: interferon alpha-2a, interferon alpha-2b, pegylated interferonalpha, including interferon alpha-2a and interferon alpha 2b, interferonbeta, interferon gamma, interferon tau, interferon omega, INFERGEN(interferon alphacon-1) by InterMune, OMNIFERON (natural interferon) byViragen, ALBUFERON by Human Genome Sciences, REBIF (interferon beta-1a)by Ares-Serono, Omega Interferon by BioMedicine, Oral Interferon Alphaby Amarillo Biosciences, and interferon gamma, interferon tau, and/orinterferon gamma-by InterMune.

In one embodiment TCN, TCN-P or a related compound and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, as disclosed herein can be used in combination oralternation with additional chemotherapeutic agents, such as thosedescribed herein or in Table 3, for the treatment of drug resistantcancer, for example multiple drug resistant cancer. Drug resistentcancers can include cancers of the colon, bone, kidney, adrenal,pancreas, liver and/or any other cancer known in the art or describedherein. In one embodiment, the additional chemotherapeutic agent can bea P-glycoprotein inhibitor. In certain non-limiting embodiments, theP-glycoprotein inhibitor can be selected from the following drugs:verapamil, cyclosporin (such as cyclosporin A), tamoxifen, calmodulinantagonists, dexverapamil, dexniguldipine, valspodar (PSC 833),biricodar (VX-710), tariquidar (XR9576), zosuquidar (LY335979),laniquidar (R101933), and/or ONT-093.

7. PHARMACEUTICAL COMPOSITIONS

The compositions including triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, can optionally be administered with a pharmaceuticalcarrier or excipient. Pharmaceutical carriers suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration. The triciribine compounds and incombination with a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, may be formulatedas the sole pharmaceutically active ingredients in the composition ormay be combined.

Compositions including the triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may be suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal, or parenteral (includingsubcutaneous, intramuscular, subcutaneous, intravenous, intradermal,intraocular, intratracheal, intracisternal, intraperitoneal, andepidural) administration. Preferably the compositions are administeredintravenously.

The compositions may conveniently be presented in unit dosage form andmay be prepared by conventional pharmaceutical techniques. Suchtechniques include the step of bringing into association one or morecompositions of the present invention and one or more pharmaceuticalcarriers or excipients.

The triciribine compounds and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, andcompositions thereof can be formulated into suitable pharmaceuticalpreparations such as solutions, suspensions, tablets, dispersibletablets, pills, capsules, powders, sustained release formulations orelixirs, for oral administration or in sterile solutions or suspensionsfor parenteral administration, as well as transdermal patch preparationand dry powder inhalers. In one embodiment, the triciribine compoundsdescribed above are formulated into pharmaceutical compositions usingtechniques and procedures well known in the art (see, e.g., Ansel,Introduction to Pharmaceutical Dosage Forms, Fourth Ed., 1985, p. 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof may be mixed with oneor more suitable pharmaceutical carriers. The compounds of the inventionmay be derivatized as the corresponding salts, esters, enol ethers oresters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids,bases, solvates, hydrates or prodrugs prior to formulation. Theconcentrations of the compounds in the compositions are effective fordelivery of an amount, upon administration, that treats, prevents, orameliorates one or more of the symptoms of the target disease ordisorder. In one embodiment, the compositions are formulated for singledosage administration. To formulate a composition, the weight fractionof compound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

Compositions suitable for oral administration may be presented asdiscrete units such as, but not limited to, tablets, caplets, pills ordragees capsules, or cachets, each containing a predetermined amount ofone or more of the compositions; as a powder or granules; as a solutionor a suspension in an aqueous liquid or a non-aqueous liquid; or as anoil-in-water liquid emulsion or a water-in-oil emulsion or as a bolus,etc.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing a triciribinecompound and optional pharmaceutical adjuvants in a carrier, such as,for example, water, saline, aqueous dextrose, glycerol, glycols,ethanol, and the like, to thereby form a solution or suspension. Ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingagents, emulsifying agents, solubilizing agents, pH buffering agents,preservatives, flavoring agents, and the like, for example, acetate,sodium citrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents. Methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

Compositions of the present invention suitable for topicaladministration in the mouth include for example, lozenges, having theingredients in a flavored basis, usually sucrose and acacia ortragacanth; pastilles, having one or more triciribine compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, of the present invention in an inertbasis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes, having one or more of the compositions of the presentinvention administered in a suitable liquid carrie

The tablets, pills, capsules, troches and the like can contain one ormore of the following ingredients, or compounds of a similar nature: abinder; a lubricant; a diluent; a glidant; a disintegrating agent; acoloring agent; a sweetening agent; a flavoring agent; a wetting agent;an emetic coating; and a film coating. Examples of binders includemicrocrystalline cellulose, gum tragacanth, glucose solution, acaciamucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone,crospovidones, sucrose and starch paste. Lubricants include talc,starch, magnesium or calcium stearate, lycopodium and stearic acid.Diluents include, for example, lactose, sucrose, starch, kaolin, salt,mannitol and dicalcium phosphate. Glidants include, but are not limitedto, colloidal silicon dioxide. Disintegrating agents includecrosscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methylcellulose, agar andcarboxymethylcellulose. Coloring agents include, for example, any of theapproved certified water soluble FD and C dyes, mixtures thereof; andwater insoluble FD and C dyes suspended on alumina hydrate. Sweeteningagents include sucrose, lactose, mannitol and artificial sweeteningagents such as saccharin, and any number of spray dried flavors.Flavoring agents include natural flavors extracted from plants such asfruits and synthetic blends of compounds which produce a pleasantsensation, such as, but not limited to peppermint and methyl salicylate.Wetting agents include propylene glycol monostearate, sorbitanmonooleate, diethylene glycol monolaurate and polyoxyethylene lauralether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, sodium carboxymethylcellulose,polyethylene glycol 4000 and cellulose acetate phthalate.

Compositions suitable for topical administration to the skin may bepresented as ointments, creams, gels, and pastes, having one or more ofthe compositions administered in a pharmaceutical acceptable carrier.

Compositions for rectal administration may be presented as a suppositorywith a suitable base including, for example, cocoa butter or asalicylate.

Compositions suitable for nasal administration, when the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, (i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose). Whenthe carrier is a liquid (for example, a nasal spray or as nasal drops),one or more of the compositions can be admixed in an aqueous or oilysolution, and inhaled or sprayed into the nasal passage.

Compositions suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining one or more of the compositions and appropriate carriers.

Compositions suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats, and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The compositions may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules, andtablets of the kind previously described above.

Pharmaceutical organic or inorganic solid or liquid carrier mediasuitable for enteral or parenteral administration can be used tofabricate the compositions. Gelatin, lactose, starch, magnesiumstearate, talc, vegetable and animal fats and oils, gum, polyalkyleneglycol, water, or other known carriers may all be suitable as carriermedia.

Compositions including triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may be used in combination with one or morepharmaceutically acceptable carrier mediums and/or excipients. As usedherein, “pharmaceutically acceptable carrier medium” includes any andall carriers, solvents, diluents, or other liquid vehicles, dispersionor suspension aids, surface active agents, isotonic agents, thickeningor emulsifying agents, preservatives, solid binders, lubricants,adjuvants, vehicles, delivery systems, disintegrants, absorbents,preservatives, surfactants, colorants, flavorants, or sweeteners and thelike, as suited to the particular dosage form desired.

Additionally, the compositions including triciribine compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, may be combined with pharmaceuticallyacceptable excipients, and, optionally, sustained-release matrices, suchas biodegradable polymers, to form therapeutic compositions. A“pharmaceutically acceptable excipient” includes a non-toxic solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type.

It will be understood, however, that the total daily usage of thecompositions will be decided by the attending physician within the scopeof sound medical judgment. The specific therapeutically effective doselevel for any particular host will depend upon a variety of factors,including for example, the disorder being treated and the severity ofthe disorder; activity of the specific composition employed; thespecific composition employed, the age, body weight, general health, sexand diet of the patient; the time of administration; route ofadministration; rate of excretion of the specific compound employed; theduration of the treatment; the triciribine compound and/or the amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, used in combination or coincidental withthe specific composition employed; and like factors well known in themedical arts. For example, it is well within the skill of the art tostart doses of the composition at levels lower than those required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved.

Compositions including triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, are preferably formulated in dosage unit form for ease ofadministration and uniformity of dosage. “Dosage unit form” as usedherein refers to a physically discrete unit of the compositionappropriate for the host to be treated. Each dosage should contain thequantity of composition calculated to produce the desired therapeuticaffect either as such, or in association with the selectedpharmaceutical carrier medium.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. For example, approximately 1-5 mg per day of acompound disclosed herein can reduce the volume of a solid tumor inmice.

The dosage will depend on host factors such as weight, age, surfacearea, metabolism, tissue distribution, absorption rate and excretionrate. In one embodiment, approximately 0.5 to 7 grams per day of atriciribine compound disclosed herein may be administered to humans.Optionally, approximately 1 to 4 grams per day of the compound can beadministered to humans. In certain embodiments 0.001-5 mg/day isadministered to a human. The therapeutically effective dose level willdepend on many factors as noted above. In addition, it is well withinthe skill of the art to start doses of the composition at relatively lowlevels, and increase the dosage until the desired effect is achieved.

Compositions including triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may be used with a sustained-release matrix, which can bemade of materials, usually polymers, which are degradable by enzymaticor acid-based hydrolysis or by dissolution. Once inserted into the body,the matrix is acted upon by enzymes and body fluids. A sustained-releasematrix for example is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (copolymers of lactic acid andglycolic acid), polyanhydrides, poly(ortho)esters, polypeptides,hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

The triciribine compounds and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, may alsobe administered in the form of liposomes. As is known in the art,liposomes are generally derived from phospholipids or other lipidsubstances. Liposomes are formed by mono- or multi-lamellar hydratedliquid crystals that are dispersed in an aqueous medium. Any non-toxic,physiologically-acceptable and metabolizable lipid capable of formingliposomes can be used. The liposome can contain, in addition to one ormore compositions of the present invention, stabilizers, preservatives,excipients, and the like. Examples of lipids are the phospholipids andthe phosphatidyl cholines (lecithins), both natural and synthetic.Methods to form liposomes are known in the art.

The triciribine compounds and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, may beformulated as aerosols for application, such as by inhalation. Theseformulations for administration to the respiratory tract can be in theform of an aerosol or solution for a nebulizer, or as a microtine powderfor insufflation, alone or in combination with an inert carrier such aslactose. In such a case, the particles of the formulation will, in oneembodiment, have diameters of less than 50 microns, in one embodimentless than 10 microns.

Compositions including the triciribine compounds and a molecule thatmodulates the HER2/neu (erbB2) receptor, for example, trastuzumab or asalt thereof, may be used in combination with other compositions and/orprocedures for the treatment of the conditions described above. Forexample, a tumor may be treated conventionally with surgery, radiation,or chemotherapy combined with one or more compositions of the presentinvention and then one or more compositions of the present invention maybe subsequently administered to the patient to extend the dormancy ofmicrometastases and to stabilize, inhibit, or reduce the growth of anyresidual primary tumor.

7.1. ADDITIONAL EMBODIMENTS

The pharmaceutical compositions including triciribine compounds and amolecule that modulates the HER2/neu (erbB2) receptor, for example,trastuzumab or a salt thereof, can be formulated according to knownmethods for preparing pharmaceutically useful compositions. Formulationsare described in a number of sources which are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa. describesformulations which can be used in connection with the subject invention.Formulations suitable for administration include, for example, aqueoussterile injection solutions, which may contain antioxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient; and aqueous and nonaqueous sterilesuspensions which may include suspending agents and thickening agents.The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in a freezedried (lyophilized) condition requiring only the condition of thesterile liquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, theformulations of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The methods of the present invention, for example, for inhibiting thegrowth of a cancerous cell, can be advantageously combined with at leastone additional therapeutic method, including but not limited tochemotherapy, radiation therapy, therapy that selectively inhibits Rasoncogenic signaling, or any other therapy known to those of skill in theart of the treatment and management of cancer, such as administration ofan anti-cancer agent.

Administration of API-2 (triciribine) as a salt may be carried out.Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids which form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting a sufficientlybasic compound such as an amine with a suitable acid affording aphysiologically acceptable anion. Alkali metal (for example, sodium,potassium or lithium) or alkaline earth metal (for example calcium)salts of carboxylic acids can also be made.

The triciribine compounds and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, can beformulated as pharmaceutical compositions and administered to a subject,such as a human or veterinary patient, in a variety of forms adapted tothe chosen route of administration, i.e., orally or parenterally, byintravenous, intramuscular, topical or subcutaneous routes.

Thus, the triciribine compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,of the present invention may be systemically administered, e.g., orally,in combination with a pharmaceutically acceptable vehicle (i.e.,carrier) such as an inert diluent or an assimilable edible carrier. Theymay be enclosed in hard or soft shell gelatin capsules, may becompressed into tablets, or may be incorporated directly with the foodof the patient's diet. For oral therapeutic administration, thecompounds may be combined with one or more excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active agent. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of the active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the compounds of the invention, sucrose or fructose as asweetening agent, methyl and propylparabens as preservatives, a dye andflavoring such as cherry or orange flavor. Of course, any material usedin preparing any unit dosage form should be pharmaceutically acceptableand substantially non-toxic in the amounts employed. In addition, thecompounds of the invention may be incorporated into sustained-releasepreparations and devices.

The triciribine compounds and a molecule that modulates the HER2/neu(erbB2) receptor, for example, trastuzumab or a salt thereof, may alsobe administered intravenously or intraperitoneally by infusion orinjection. Solutions of the active agents or their salts can be preparedin water, optionally mixed with a nontoxic surfactant. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, triacetin,and mixtures thereof and in oils. Under ordinary conditions of storageand use, these preparations contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powdersincluding the active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform must be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium including, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theuse in the compositions of agents delaying absorption, for example,aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating compounds ofthe invention in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the triciribine compounds and a moleculethat modulates the HER2/neu (erbB2) receptor, for example, trastuzumabor a salt thereof, may be applied in pure-form, i.e., when they areliquids. However, it will generally be desirable to administer them tothe skin as compositions or formulations, in combination with adermatologically acceptable carrier, which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the compounds of the invention can be dissolved ordispersed at effective levels, optionally with the aid of non-toxicsurfactants. Adjuvants such as fragrances and additional antimicrobialagents can be added to optimize the properties for a given use. Theresultant liquid compositions can be applied from absorbent pads, usedto impregnate bandages and other dressings, or sprayed onto the affectedarea using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the compounds of the invention to the skinare disclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S.Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman(U.S. Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

In one non-limiting embodiment, the concentration of the active agent ina liquid composition, such as a lotion, can be from about 0.1-25 wt-%,or from about 0.5-10 wt.-%. In one embodiment, the concentration in asemi-solid or solid composition such as a gel or a powder can be about0.1-5 wt.-%, preferably about 0.5-2.5 wt.-%. In one embodiment, singledosages for injection, infusion or ingestion will generally vary between5-1500 mg, and may be administered, i.e., 1-3 times daily, to yieldlevels of about 0.1-50 mg/kg, for adults. A non-limiting dosage of thepresent invention can be between 7.5 to 45 mg per clay, administeredorally, with appropriate adjustment for the body weight of anindividual.

Accordingly, the present invention includes a pharmaceutical compositionincluding triciribine compounds and a molecule that modulates theHER2/neu (erbB2) receptor, for example, trastuzumab or a salt thereof,or pharmaceutically acceptable salts thereof, in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, including an amount oftriciribine compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab or a salt thereof, or apharmaceutically acceptable salt thereof, constitute a preferredembodiment of the invention. The dose administered to a subject,particularly a human, in the context of the present invention should besufficient to affect a therapeutic response in the patient over areasonable time frame. One skilled in the art will recognize that dosagewill depend upon a variety of factors including the condition of theanimal, the body weight of the animal, as well as the severity and stageof the cancer.

A suitable dose is that which will result in a concentration of thetriciribine compounds and a molecule that modulates the HER2/neu (erbB2)receptor, for example, trastuzumab, in tumor tissue which is known toaffect the desired response. The preferred dosage is the amount whichresults in maximum inhibition of cancer cell growth, withoutunmanageable side effects. Administration of API-2 (or apharmaceutically acceptable salt thereof) can be continuous or atdistinct intervals, as can be determined by a person of ordinary skillin the art.

Mammalian species which benefit from the disclosed methods for theinhibition of cancer cell growth, include, but are not limited to,primates, such as apes, chimpanzees, orangutans, humans, monkeys;domesticated animals (e.g., pets) such as dogs, cats, guinea pigs,hamsters, Vietnamese pot-bellied pigs, rabbits, and ferrets;domesticated farm animals such as cows, buffalo, bison, horses, donkey,swine, sheep, and goats; exotic animals typically found in zoos, such asbear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros,giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs,koala bears, kangaroo, opossums, raccoons, pandas, hyena, seals, sealions, elephant seals, otters, porpoises, dolphins, and whales. Theterms “patient” and “subject” are used herein interchangeably and areintended to include such human and non-human mammalian species.Likewise, in vitro methods of the present invention can be earned out oncells of such mammalian species.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical profession.

The following examples are offered by way of illustration and not by wayof limitation.

8. EXAMPLES Example 1 In Vitro Screening

Cell Lines and NCI Diversity Set. All cell lines can be purchased fromATCC or described previously (Cheng J. Q. et al., Oncogene, 1997. 14: p.2793-2801; West, K. A. et al., Drug Resist Updat, 2002. 5: p. 234-248;Satyamoorthy K. et al., Cancer Res, 2001. 61: p. 7318-7324). The NCIStructural Diversity Set is a library of 1, 992 compounds selected fromthe approximately 140,000-compound NCI drug depository. In-depth data onthe selection, structures, and activities of these diversity setcompounds can be found on the NCI Developmental Therapeutics Program website.

Screening for Inhibition of Akt-Transformed Cell Growth.

AKT2 transformed NIH3T3 cells or LXSN vector-transfected NIH3T3 controlcells (Cheng J. Q. et al., Oncogene, 1997. 14: p. 2793-2801) are platedinto 96-well tissue culture plate. Following treatment with 5 μM of NClDiversity Set compound, cell growth can be detected with CellTier 96 OneSolution Cell Proliferation kit (Promega). Compounds that inhibit growthin AKT2-transformed but not LXSN-transfected NIH3T3 cells are consideredas candidates of Akt inhibitor and subjected to further analysis.

In Vitro Protein Kinase

Cell Survival and Apoptosis Assays. In vitro kinase is performed aspreviously described (see, for example, Jiang K. et al., Mol Cell Biol,2000. 20: p. 139-148). Cell survival is assayed with MTS (Promega).Apoptosis was detected with annexin V, which is performed as previouslydescribed (Jiang K. et al., Mol Cell Biol, 2000. 20: p. 139-148).Recombinant Akt and PDK1 are purchased from Upstate Biotechnology Inc.

Results

Identification of Small Molecule Inhibitor of Akt Signaling Pathway,API-2.

Frequent alterations of Akt has been detected in human cancer anddisruption of Akt pathway induces apoptosis and inhibits tumor growth(Jetzt A. et al., Cancer Res, 2003. 63: p. 697-706, 2003). Thus, Akt isconsidered as an attractive molecular target for development of novelcancer therapeutics. To identify small molecule inhibitor(s) of Akt, achemical library of 1,992-compounds from the NCl (the NCl Diversity Set)is evaluated for agents capable of inhibition of growth inAKT2-transformed but not empty vector LXSN-transfected NIH3T3 cells.Repeated experiments showed that 32 compounds inhibited growth only inAKT2-transformed cells. The most potent of these compounds, API-2 (NClidentifier: NSC 154020), can suppress cell growth at a concentration of50 nM. FIG. 1A shows the chemical structure of API-2, which is alsoknown as triciribine (Schweinsberg P. D. et al., Biochem Pharmacol,1981. 30: p. 2521-2526). The fact that API-2 inhibits selectively AKT-2transformed cells over untransformed parental cells prompted us todetermine whether API-2 is an inhibitor of AKT2 kinase. To this end,AKT2 is immunoprecipitated with anti-AKT2 antibody from AKT-2transformed NIH3T3 cells following treatment with API-2. AKT2immunoprecipitates were immunoblotted with anti-phospho-Akt antibodies.As shown in FIG. 1B, API-2 significantly inhibited AKT2 phosphorylationat both threonine-309 and serine-474, which are required for fullactivation of AKT2 (Datta S. R. et al., Genes Dev, 1999. 13: p.2905-2927). As three isoforms of Akt share high homology and similarstructure, the effect of API-2 on their kinase activities is evaluated.HEK293 cells are transfected with HA-Akt1, -AKT2 and -AKT3,serum-starved overnight and treated with API-2 for 60 min prior to EGF(50 ng/ml) stimulation. Triple experiments showed that API-2 suppressedEGF-induced kinase activity and phosphorylation of Akt1, AKT2 and AKT3(FIG. 1C). However, kinase activity of recombinant constitutively activeAKT2 (Myr-AKT2) is not inhibited by API-2 in an in vitro kinase reaction(FIG. 1D), suggesting that API-2 does not directly inhibit Akt in vitroand that API-2 neither functions as ATP competitor nor as the substratecompetitor that binds to active site of Akt.

API-2 does not Inhibit Known Upstream Activators of Akt.

It has been well documented that Akt is activated by extracellularstimuli and intracellular signal molecules, such as active Ras and Src,through a PI3K-dependent manner. Therefore, API-2 inhibition of Aktcould result from targeting upstream molecule(s) of Akt. As PI3K andPDK1 are direct upstream regulators of Akt (Datta S. R. et al., GenesDev, 1999. 13: p. 2905-2927), whether API-2 inhibits PI3K and/or PDK1 isexamined HEK293 cells are serum-starved and then can be treated withAPI-2 or PI3K inhibitor, wortmannin, for 30 min prior to EGFstimulation. PI3K is immunoprecipitated with anti-p110 antibody. Theimmunoprecipitates are subjected to in vitro PI3K kinase assay usingPI-4-P as a substrate. As shown in FIG. 2A, the EGF-induced PI3Kactivity is inhibited by wortmannin but not by API-2. To evaluate theeffect of API-2 on PDK1, an assay in which recombinant PDK1 promotes thethreonine-309 phosphorylation of AKT2 peptides is used in the presenceof lipid vesicles containing phosphotidylinositol. As shown in FIG. 2B,the assay is potently inhibited by the control PDK1 inhibitorstaurosporine (IC50=5 nM). In contrast, API-2 displayed only 21%inhibition of the assay at the highest concentration tested (5.1 μM). Tofurther evaluate the effect of API-2 on PDK1 activation, theautophosphorylation level of PDK1 at serine-241, a residue that isphosphorylated by itself and is critical for its activity is examined(Datta S. R. et al., Genes Dev, 1999. 13: p. 2905-2927), following API-2treatment of HEK293 cells. Triplicate experiments show thatphosphorylation levels of PDK1 are not inhibited by API-2 (FIG. 2B).However, PI3K inhibitor wortmannin can inhibit EGF-stimulated PDK1 (FIG.2B).

API-2 is Highly Selective for the Akt Over PKC, PKA, SGK, STAT, JNK,p38, and ERK Signaling Pathways.

Akt belongs to AGC (PKA/PKG/PKC) kinase family, which also include PKA,PKC, serum- and glucocorticoid-inducible kinase (SGK), p90 ribosomal S6kinase, p70^(S6K) mitogen- and stress-activated protein kinase andPKC-related kinase. Among AGC kinase family, protein structures of PKA,PKC and SGK are more close to Akt kinase than other members. Therefore,next examined are the effects of API-2 on the enzymatic activities ofthese 3 kinases. HEK293 cells are transfected with HA-tagged PKA, PKCα,or SGK. In vitro kinase assay and immunoblotting analysis show that thekinase activities of PKA and PKC are inhibited by PKAI and Ro 31-8220, aPKC inhibitor, respectively, whereas API-2 exhibits no effect on theiractivities (FIGS. 2C and 2E). Further, serum-induced SGK kinase activityis attenuated by wortmannin but not by API-2 (FIG. 2D). In addition, itis determined whether API-2 has effect on other oncogenic survivalpathways. Western blotting analyses with commercially availableanti-phospho-antibodies reveals that phosphorylation levels of Stat3,JNK, p38 and Erk1/2 were not affected by API-2 treatment (FIG. 2F).These data indicate that API-2 specifically inhibits Akt signalingpathway.

API-2 Suppresses Cell Growth and Induces Apoptosis inAkt-Overexpressing/Activating Human Cancer Cell Lines.

The ability of API-2 to selectively inhibit the Akt pathway suggeststhat it should inhibit proliferation and/or induces apoptosispreferentially in those tumor cells with aberrant expression/activationof Akt. As activation of Akt in human malignancies commonly results fromoverexpression of Akt or PTEN mutations, API-2 is used to treat thecells that express constitutively active Akt, caused by overexpressionof AKT2 (OVCAR3, OVCAR8, PANC1 and AKT2-transformed NIH3T3) or mutationsof the PTEN gene (PC-3, LNCaP, MDA-MB-468), and cells that do not(OVCAR5, DU-145, T47D, COL0357 and LXSN-NIH3T3) as well as melanomacells that are activated by IGF-1 to activate Akt or do not respond togrowth stimulation by IGF-1 (Satyamoorthy K. et al., Cancer Res, 2001.61: p. 7318-7324) Immunoblotting analysis showed that phosphorylationlevels of Akt are inhibited by API-2 only in the cells expressingelevated Akt or responding to IGF-1 simulation (FIG. 3A). Accordingly,API-2 inhibited cell growth to a much higher degree inAkt-overexpressing/activating cells as compared to those with low levelsof Akt. As shown in FIG. 3B, API-2 treatment inhibited cellproliferation by approximately 50-60% in Akt-overexpressing/activatingcell lines, LNCaP, PC-3, OVCAR3, OVCA8, PANC1, MDA-MB-468, and WM35,whereas only by about 10-20% in DU145, OVCAR5, COL0357, T47D and WM852cells, which exhibit low levels of Akt or do not respond to growthstimulation by IGF-1. Moreover, API-2 induces apoptosis by 8-fold(OVCAR3), 6-fold (OVCAR8), 6-fold (PANC1), and 3-fold (AKT2-NIH3T3). Nosignificant difference of apoptosis is observed between API-2 andvehicle (DMSO) treatment in OVCARS, COL0357 and LXSN-NIH3T3 cells. Thus,API-2 inhibits cell growth and induces apoptosis preferentially in cellsthat express aberrant Akt.

API-2 Inhibits Downstream Targets of Akt.

It has been shown that Akt exerts its cellular effects throughphosphorylation of a number of proteins (Datta S. R. et al., Genes Dev,1999. 13: p. 2905-2927). More than 20 proteins have been identified asAkt substrates, including the members of Forkhead protein family (FKHR,AFX and FKHRL1), tuberlin/TSC2, p70^(S6K), GSK-3, p21^(WAFI/Cip1),p27^(kip1), MDM2, Bad, ASK1 and IKK etc. It is next examined whetherAPI-2 inhibits downstream targets of Akt. As anti-phospho-tuberlin,-Bad, -AFX, and -GSK-3β antibodies are commercially available,therefore, the effect of API-2 on their phosphorylation induced by Aktwas determined Following API-2 (1 μM) treatment, OVCAR3 cells was lysedand immunoblotted with the individual anti-phospho-antibody. FIG. 4Ashows that API-2 considerably inhibited the phosphorylation levels oftuberlin leading to stabilization and upregulation of tuberin (Dan, H.C., et al., J Biol Chem, 2002. 277: p. 35364-35370). The phosphorylationlevels of Bad, GSK-3β, and AFX are partially attenuated by API-2. Thesedata suggest that API-2 induces cell death and cell growth arrest byinhibiting phosphorylation of its downstream targets. API-2 inhibitionof Akt downstream targets at different degrees could be due to the factthat phosphorylation sites of these targets are also regulated by otherkinase(s), for instance, Bad serine-136 is phosphorylated by PAK1 inaddition to Akt (Schurmann, A., et al. Mol Cell Biol, 2000. 20: p.453-461).

Example 2 Antitumor Activity in the Nude Mouse Tumor Xenograft Model

Tumor cells can be harvested, suspended in PBS, and can be injected s.c.into the right and left flanks (2×10⁶ cells/flank) of 8-week-old femalenude mice as reported previously (Sun, J. et al., Cancer Res, 1999. 59:p. 4919-4926, 1999). When tumors reach about 100-150 mm³, animals arerandomized and dosed i.p. with 0.2 ml vehicle of the triciribinecompound and/or a molecule that modulates the HER2/neu (erbB2) receptor,for example, trastuzumab, daily. Control animals receive DMSO (20%)vehicle, whereas treated animals can be injected with API-2 (1mg/kg/day) in 20% DMSO.

API-2 Inhibits the Growth of Tumors in Nude Mice that Overexpress Akt.

Frequent overexpression/activation and/or amplification of AKT1 and AKT2in human ovarian and pancreatic cancer was shown (Cheng J. Q. andNicosia S. V., in Encyclopedic Reference of Cancer, 2001, Schwab D, Ed.Springer, Berlin Heidelberg and New York, p. 35-7) Inhibition of Aktpathway by inhibitors of PI3K, HSP70, Src and farnesyltransferaseresulted in cell growth arrest and induction of apoptosis (Solit D. B.et al., Cancer Res, 2003. 63: p. 2139-2144; Xu, W., et al. Cancer Res,2003.63: p. 7777-7784). A recent study showed that the tumor growth ofxenografts with elevated Akt was also significantly inhibited byintratumoral injection of adenovirus of dominant negative Akt (Jetzt A.et al., Cancer Res, 2003. 63: p. 697-706, 2003). Because API-2 inhibitsAkt signaling and induces apoptosis and cell growth arrest only incancer cells with elevated levels of Akt (FIG. 3), the growth of tumorswith elevated levels of Akt should be more sensitive to API-2 than thatof tumors with low levels of Akt in nude mice. To this end, s.c.Akt-overexpressing cells (OVCAR3, OVCAR8 and PANC-1) are s.c. implantedinto the right flank, and those cell lines that express low levels ofAkt (OVCARS and COL0357) into the left flank of mice. When the tumorsreach an average size of about 100-150 mm³, the animals are randomizedand treated i.p. with either vehicle or API-2 (1 mg/kg/day). Asillustrated in FIG. 4B, OVCAR-5 and COLO357 tumors treated with vehiclegrew to about 800-1,000 mm³ 49 days after tumor implantation. OVCAR3,OVCAR8 and PANC1 tumors treated with vehicle control grew to about700-900 mm³ 49 days after tumor implantation. API-2 inhibited OVCAR3,OVCAR8 and PANC1 tumor growth by 90%, 88% and 80%, respectively. Incontrast, API-2 has little effect on the growth of OVCAR5 and COL0357cells in nude mice (FIGS. 4B-4D and data not shown). At dose 1mg/kg/day, API-2 had no effects on blood glucose level, body weight,activity and food intake of mice. In treated tumor samples, Akt activitywas inhibited by API-2 without change of total Akt content (FIG. 4E).Taken together, these results indicate that API-2 selectively inhibitsthe growth of tumors with elevated levels of Akt.

Example 3 TCN Directly Inhibits Wild Type Akt Kinase Activity

API-2 (TCN) can directly inhibit wild type Akt kinase activity inducedby PDK1 in vitro (FIG. 1). This result supports that API-2 is a directAkt inhibitor and that the underlying mechanism may be API-2 binding toPH domain and/or threonine-308 of Akt. An in vitro kinase assay isperformed with recombinant of PDK1 and Akt in a kinase buffer containingphosphatidylinositol-3,4,5-P3 (PIP3), API-2 and histone H2B assubstrate. After incubation of 30 min, the reactions were separated bySDS-PAGE and exposed in a film.

Example 4 TCN is Effective in Cancer Resistant Cells

The effects of TCN (API-2) are tested in cisplatin, paclitaxel, andtamoxifen resistant A270CP, C-13, OVCAR433 and MCF7/TAM cells. API-2overcame cisplatin, paclitaxel, and tamoxifen resistance in these cells.

Example 5 TCN Potentiates Growth Inhibition by Trastuzumab and InducesApoptosis

Materials and Methods

Cell Lines and Cell Cultures.

The tumorigenic BT474.m1 subline maintained in Dulbecco's modifiedEagle's medium: Ham's F-12 medium (1:1) with 8-10% FBS.

Antibodies and Reagents.

Trastuzumab was a gift from Genentech (San Francisco, Calif.). RAD001(everolimus) was a gift from Novartis (East Hanover, N.J.). QLT0267 andKP 372-1 were gifts from QLT Inc. (Vancouver, BC). Triciribine(6-Amino-4-methyl-8-((3-D-ribofuranosyl)-4H,8H-pyrrolo[4,3,2-de]pyrimido[4,5-c]pyridazine)was purchased from Berry & Associates, Inc. (Ann Arbor, Mich.).Edelfosine was purchased from Calbiochem (San Diego, Calif.). Aselective Akt inhibitor, 4ADPIB(4-amino-2(3,4-dichloro-phenyl)-N-(1H-indazol-5-yl)-butyramide) (U.S.Pat. No. 6,919,340), was synthesized. PTEN antibodies were from SantaCruz Biotechnology (Santa Cruz, Calif.). 3-actin antibodies were fromSigma (St. Louis, Mo.). All other antibodies were purchased from CellSignaling Technology (Danvers, Mass.).

PTEN Antisense and Non-Specific Oligonucleotide Transient Transfection.

Antisense (AS) oligonucleotides specific for PTEN, control non-specific(NS) oligonucleotides and procedures for transfection were followedaccording to Nagata Y. et al., Cancer Cell, 2004. 6(2): p. 117-27.

Cell Proliferation Assay.

PTEN AS/NS transfected BT474.m1 cells were plated 2500 cells/0.32 cm²well. Cells were treated with inhibitors of the Akt/mTOR pathway aloneor in combination with trastuzumab as described for 5 days and viablecells were measured by MTS assay using the CellTiter 96 AQnonradioactive cell proliferation assay kits according to themanufacturer's protocol (Promega, Madison, Wis.). Treated cells werecompared to control DMSO treated BT474.m1 cells to calculate percentageof growth inhibition.

APO-BRDU TUNEL Assay.

The PTEN AS and NS transfected BT474.m1 cells were plated in 6-wellplates (4-6×10⁵ cells/well). Twenty-four hours after plating, the cellswere treated as indicated for 72 hours with trastuzumab, TCN and/orRAD001. The floating and adherent cells were collected, labeled andstained using the APO-BRDU™ TUNEL assay kit (Phoenix Flow Systems, SanDiego, Calif.) according to the manufacturer's protocol. Data wascollected and analyzed using a FACScan flow cytometer and CellQuest Pro4.02 software (Becton Dickinson, Franklin Lakes, N.J.). At least 10,000events were examined

SDS-PAGE and Immunoblot Analysis.

Cells transfected with PTEN AS/NS oligonucleotides were treated asindicated. Immunoblotting was performed as described by Nagata Y. etal., Cancer Cell, 2004. 6(2): p. 117-27.

Results

Triciribine and RAD001 Potentiate Growth Inhibition by Trastuzumab inPTEN-Deficient Cells.

To find a strategy to overcome trastuzumab resistance, particularlyresistance caused by PTEN loss, 6 different small molecule inhibitorswere tested, which directly or indirectly targeted the PI3K/Akt/mTORsignal transduction pathway, a major pathway activated by overexpressionof ErbB2 and the loss of PTEN. The goal was to identify compounds thatwould exhibit synergistic effects with trastuzumab, preferably at a lowdose of the compound in order to minimize toxicity. The drugs chosentargeted Akt, mTOR and integrin-linked kinase (ILK) (FIG. 9A). BT474.m1cells are a tumorigenic subline of the BT474 breast cancer cell line andexpress high levels of ErbB2. When PTEN levels are decreased bytransfection with PTEN antisense oligonucleotides (PTEN AS), BT474.m1breast cancer cells become more resistant to the anti-proliferativeeffects of trastuzumab than cells with normal levels of PTEN and providea good experimental model for breast cancers in which trastuzumabresistance is caused by PTEN loss (Nagata Y. et al., Cancer Cell, 2004.6(2): p. 117-27 and FIGS. 9B and 9C). Nonspecific oligonucleotides (NS)were transfected as controls. Treatment with PTEN AS oligonucleotideseffectively lowered PTEN levels (FIGS. 11A and 11B). Neither PTEN AS norNS control oligonucleotides altered ErbB2 levels in the cells.

PTEN AS and NS-transfected BT474.m1 cells were treated with each of the6 compounds or trastuzumab alone and in combination for 5 days andevaluated cell proliferation as compared to DMSO-treated control. Usinggrowth inhibition as a biological endpoint, we compared the ability ofeach drug to exhibit cooperative effects with trastuzumab using doses ofdrug that resulted in −20-40% growth inhibition when administered alone(FIG. 9A).

Almost all of the compounds displayed growth inhibitory effects,particularly at high concentrations and in cells with intact PTEN (FIG.9A). However, two of the compounds, triciribine and RAD001, markedlyenhanced growth inhibition in the PTEN AS cells when combined withtrastuzumab as compared to trastuzumab or either compound alone (FIG.9A). Triciribine (also called API-2), a compound that inhibits Aktactivation, potentiated growth inhibition by trastuzumab over a 20-foldconcentration range (FIG. 9B). The mTOR inhibitor RAD001 (everolimus)increased growth inhibition by trastuzumab when RAD001 was administeredat low doses (<1 nM) (FIG. 9C). Strikingly, triciribine and RAD001 wereable to cooperate with trastuzumab to inhibit cell growth at similarlevels in the PTEN AS and NS cells (FIGS. 9B and 9C). In essence,triciribine and RADOO1 were able to restore trastuzumab sensitivity toPTEN-deficient cells. Triciribine and RADOO1 were also effective assingle agents, both in PTEN AS and NS cells, at doses greater than 5 mMand 1.5 nM for triciribine and RAD001, respectively (FIGS. 9B and 9C).

A third compound, the ILK inhibitor QLT0267, potentiated growthinhibition by trastuzumab within a narrow dose range (−5-15 pM)(FIG.9A). Because the dose range in which QLT0267 exhibited cooperativeeffects with trastuzumab was narrow, this compound further was notinvestigated further. At concentrations greater than 20 pM, QLT0267 hadno cooperative effect with trastuzumab but significantly inhibited cellgrowth as a single agent.

Induction of Apoptosis Following Combination Treatment.

To assess if growth inhibition was accompanied by apoptosis, we treatedPTEN AS and NS transfected BT474.m1 cells with triciribine, RAD001 andtrastuzumab, alone or combined, and quantified the levels of apoptosis(FIG. 10). RAD001 did not significantly induce apoptosis alone or incombination with trastuzumab. Although the number of TUNEL-positive,apoptotic cells increased slightly following treatment with trastuzumabor triciribine alone, this increase was not statistically significant.However, the combination of triciribine and trastuzumab, significantlyinduced apoptosis as compared with all other treatments in both PTEN ASand NS transfected cells (FIG. 10).

Example 6 TCN Inhibits Activation of Akt and mTOR Inhibition ofDownstream Signaling Molecules

Immunoblot analysis verified that triciribine and RAD001 blockedactivation of Akt and mTOR, important signaling molecules activated byErbB2 and the targets of triciribine and RAD001 respectively.Phosphorylation of Akt on Thr308 and Ser473 was analyzed as an indicatorof Akt activity and mTOR activity was assessed by the phosphorylation ofp70S6K (70-kDa ribosomal protein S6 kinase), an mTOR target. Aftertriciribine treatment, phosphorylation of Akt on both sites wassubstantially decreased (FIG. 11A). In PTEN-deficient cells, the levelsof Akt phosphorylation following triciribine and trastuzumab combinationtreatment were similar to those seen in cells with intact PTEN (FIG.11A, lanes 4&8). Thus, triciribine overcame the adverse effects of PTENloss by effectively blocking Akt activation. RAD001 dramatically blockedphosphorylation of p70S6K (FIG. 11B). However, RAD001 combined withtrastuzumab did not lower p70S6K phosphorylation beyond that seen withRAD001 alone (FIG. 11B). A feedback loop has been recently identifiedwhich results in Akt phosphorylation and activation following treatmentwith mTOR inhibitors, such as RAD001 (O'Reilly K. E. et al., Cancer Res,2006. 66(3): p. 1500-8). It was also observed that feedback activationof Akt by RAD001 and combination therapy with trastuzumab and RAD001eliminated Akt phosphorylation by this feedback loop (FIG. 11B, lanes 3vs. 4), consistent with the notion that Akt activation following mTORinhibition is dependent on upstream receptor tyrosine kinases (O'ReillyK. E. et al., Cancer Res, 2006. 66(3): p. 1500-8). In summary, bothdrugs inhibited their predicted target kinases and combination treatmenthad a larger inhibitory effect on the Akt/mTOR signaling pathway thanany single agent, even in PTEN-deficient cells.

Example 7 TCN and Trastuzumab Inhibit Tumor Growth in PTEN-DeficientTumors

Materials and Methods

Xenograft Human Tumor Model in SCID Mice.

Female, 6-week-old, severe combined immunodeficiency (SCID) mice werefrom Taconic Farms (Hudson, N.Y.). Tumor xenografts were performed asdescribed in Nagata Y. et al., Cancer Cell, 2004. 6(2): p. 117-27. Whenthe xenograft tumors reached the average size of 100-150 mm³, the micewere divided into 6 groups, each with 7 mice and an even distribution oftumor sizes, and treated as follows. PTEN antisense (30 pg)oligonucleotides were administered to each mouse weekly via intratumorinjection. One week after PTEN AS oligonucleotide administration wasinitiated, drug treatment began. Trastuzumab was given at a dose of 0.5mg/kg twice a week in 200 pL saline through intratumor injection atmultiple sites. Triciribine was given at a dose of 0.5 mg/kg/day in 200pL 20% DMSO saline solution through intraperitoneal (I.P.) injection.RAD001 was given via gavage at a dose of 1 pg/kg in 500 pL 5% glucosewater twice a week. 20% DMSO saline solution (200 pL/day) was giventhrough I.P. injection. The tumors were measured twice weekly withcalipers and the volume of the tumors was calculated as:volume=length×width/2.

Statistical Analysis.

One-way ANOVA was performed using GraphPad Prism 3.0 for Windows (GraphPad Software, San Diego, Calif.).

Results

The earlier biological and molecular data were very promising, however,in vivo studies provide the most stringent test for therapeuticefficacy. Therefore, triciribine and RAD001 were tested in vivo.BT474.m1 cell xenografts were injected into the mammary fat pad of6-week-old SCID mice. After tumors formed, the mice received PTEN ASweekly via intratumor injection. This protocol effectively modelsPTEN-deficient tumors in vivo (Nagata Y. et al., Cancer Cell, 2004.6(2): p. 117-27). The mice were randomized into treatment groupsreceiving triciribine, RAD001, trastuzumab or DMSO alone or incombination. After treatment, the growth patterns of the tumors treatedwith DMSO, trastuzumab, RAD001, or triciribine alone were similar (FIGS.12A and 12B). Growth of the tumors was not inhibited and the mice wereeuthanized after 3 weeks due to large tumor burdens. In contrast,combination treatment with triciribine and trastuzumab dramatically andsignificantly inhibited tumor growth (FIG. 12A). Many of the tumorsactually decreased in size and four of 7 mice had no palpable tumorsafter 5 weeks of treatment. Following treatment with RAD001 andtrastuzumab, tumor growth was relatively slower compared to RAD001 ortrastuzumab alone (FIG. 12B). Thus combining trastuzumab withtriciribine or RAD001 effectively inhibited ErbB2-overexpressing,PTEN-deficient human breast cancer xenografts in vivo.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. It is intended that all of thesevariations and modifications be included within the scope of thisinvention.

1.-27. (canceled)
 28. A method for treating a subject having a tumor orcancer, which tumor or cancer overexpresses AKT kinase comprising: (i)determining whether the subject has a tumor or cancer that overexpressesAKT kinase; (ii) if said subject has a tumor or cancer thatoverexpresses AKT kinase, a. administering to said subject: i. at leastone compound of formula I selected from the group consisting of thefollowing compounds:

wherein each R₂′, R₃′, and R₅′ is independently hydrogen; optionallysubstituted phosphate or phosphonate, mono-, di-, or triphosphate; acyl;lower acyl; alkyl; lower alkyl; amide; sulfonate ester; alkyl sulfonateester; arylalkyl sulfonate ester; sulfonyl; methanesulfonyl; benzylsulfonyl, wherein the phenyl group of said benzyl is optionallysubstituted with one or more halo, hydroxyl, amino, alkylamino,arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate,phosphonic acid, phosphate, or phosphonate; optionally substitutedarylsulfonyl; a lipid; phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup that, in vivo, provides a compound of said formula I wherein R₂′,R₃′ or R₅′ is independently H or mono-, di- or tri-phosphate; whereinR^(x) and R^(y) are independently hydrogen; optionally substitutedphosphate; acyl; lower acyl; amide; alkyl; lower alkyl; aromatic;polyoxyalkylene; polyethyleneglycol; optionally substitutedarylsulfonyl; a lipid; a phospholipid; an amino acid; a carbohydrate; apeptide; or cholesterol; or other pharmaceutically acceptable leavinggroup; and wherein R₁ and R₂ each are independently H, optionallysubstituted straight chained, branched or cyclic alkyl, lower alkyl,alkenyl, or alkynyl, CO-alkyl, CO-alkenyl, CO-alkynyl, CO-aryl orheteroaryl, CO-alkoxyalkyl, CO-aryloxyalkyl, CO-substituted aryl,sulfonyl, alkylsulfonyl, arylsulfonyl, or aralkylsulfonyl; ii.trastuzumab or a salt thereof; and iii. a pharmaceutically acceptablecarrier.
 29. The method of claim 28, wherein the compound of formula Iis triciribine.
 30. The method of claim 28, wherein the compound offormula I is triciribine phosphate.
 31. The method of claim 28, whereinthe compound of formula I is triciribine phosphonate.
 32. The method ofclaim 28, wherein the compound of formula I is present in a dose amountof at least 20 mg/m².
 33. The method of claim 28, wherein the compoundof formula I is present in an amount of at least 10 mg/m².
 34. Themethod of claim 28, wherein the administration is parenteraladministration.
 35. The method of claim 34, wherein the parenteraladministration is intravenous administration.
 36. The method of claim28, wherein the administration is oral administration.
 37. The method ofclaim 28, wherein the one or more platinum compounds is present in adose about 0.1 mg/m² to about 200 mg/m².
 38. The method of claim 28,wherein the trastuzumab or salt thereof is present in a dose about 1mg/m² to about 150 mg/m².
 39. The method of claim 28, wherein thetrastuzumab or salt thereof is present in a dose about 10 mg/m² to about100 mg/m².
 40. The method of claim 28, wherein the trastuzumab or saltthereof is present in a dose about 25 mg/m² to about 50 mg/m².
 41. Themethod of claim 28, wherein the administration of a compound of formulaI and the trastuzumab or salt thereof is concurrently administered. 42.The method of claim 28, wherein the administration of a compound offormula I is followed by the administration of the trastuzumab or saltthereof.
 43. The method of claim 28, wherein the administration of thetrastuzumab or salt thereof is followed by the administration of acompound of formula I.